MHC class II deficiency patients are mutated for transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. Four complementation groups (A-D) are defined and the gene defective in group A has been shown to encode the MHC class II transactivator (CIITA). Here, we report the molecular characterization of a new MHC class II deficiency patient, ATU. Cell fusion experiments indicated that ATU belongs to complementation group A. Subsequent mutation analysis revealed that the CIITA mRNA lacked 84 nucleotides. This deletion was the result of the absence of a splice donor site in the CIITA gene of ATU. As a result of this novel homozygous genomic deletion, ATU CIITA failed to transactivate MHC class II genes. Furthermore, this truncated CIITA of ATU did not display a dominant negative effect on CIITA-mediated transactivation of various isotypic MHC class II promoters.

The major histocompatibility complex (MHC) class II plays a key role in adaptive immunity by presenting foreign peptides to CD4(+) T cells and by triggering the adaptive immune response. While the structure and function of MHC class II have been well characterized in mammalian, limited research has been done on fishes. In this study, we characterized the gene structure and expression of MHC class II α (Lunar-DAA) and II β (Lunar-DAB) of mangrove red snapper (Lutjanus argentimaculatus). Both genes shared, respectively, a high similarity and typical features with other vertebrate MHC class II α and II β. The phylogenetic analysis of the deduced peptides revealed that both Lunar-DAA and Lunar-DAB were located in the teleost subclass. Western blotting analyses indicated that both MHC class II α and II β were expressed ubiquitously in immune-related cells, tissues and organs, and that MHC class II α and II β chains existed mainly as heterodimers. While it was highly expressed in gills, thymus, head kidney (HK), spleen, head kidney macrophage and spleen leucocytes, MHC class II β chain was expressed with a low abundance in skin, intestine, stomach and heart. The highest expression of MHC class II β in thymus confirmed the conclusion that thymus is one of the primary lymphoid organs in fishes. The detection of MHC class II αβ dimers in HK macrophages and spleen leucocytes indicated that HK macrophages and spleen leucocytes play a critical role in the adaptive immunity in fishes. All these results provide valuable information for understanding the structure of MHC class II α and II β and their function in immune responses.

The ability of chronically demyelinated central nervous system (CNS) tissue to express major histocompatibility complex (MHC) class II molecules has been measured in mouse spinal cord cultures exposed for 1 and 3 weeks to demyelinating anti-white matter (WM) serum. From previous studies, It was known that after 3 weeks of demyelination in vitro, such cultures are incapable of remyelination. In the present report, MHCII levels were evaluated by immunocytochemistry and by Western and Northern blots. The results have shown that after both 1 and 3 weeks of exposure to myelinotoxic anti-WM serum, the cultures retained the ability to expressMHCII and this could be further upregulated by incubation with interferon {gamma} (IFN{gamma}). Control groups showed increased expression of MHCII with age. By immunocytochemistry, all groups of cultures expressed high levels of MHCII and all groups showed upregulation after IFN{gamma} treatment. Anti-WM-treated cultures demonstrated slightly higher levels of MHCII than controls. Morphologically, the MHCIIexpression was associated with the surface of astrocytes. Semiquantitative analysis by Western blotting confirmed the increase in class IIMHCexpression in the long-term treated cultures after IFN{gamma} exposure, revealing no differences between anti-WM-treated and complement-treated cultures. This was also supported by Northern blotting which showed similar mRNA levels in both groups. These findings suggest that long-term demyelinated CNS tissue still possesses the ability to interact with CD4{sup +} T cells, observations of significance to the expansion of the chronic multiple sclerosis lesion. 50 refs., 6 figs., 2 tabs.

Adaptive immunity, which plays an important role in the development of atherosclerosis, is mediated by major histocompatibility complex (MHC)-dependent antigen presentation. In atherosclerotic lesions, macrophages constitute an important class of antigen-presenting cells that activate adaptive immune responses to oxidized low-density lipoprotein (OxLDL). It has been reported that autophagy regulates adaptive immune responses by enhancing antigen presentation to MHC class II (MHC-II). In a previous study, we have demonstrated that SYK (spleen tyrosine kinase) regulates generation of reactive oxygen species (ROS) and activation of MAPK8/JNK1 in macrophages. Because ROS and MAPK8 are known to regulate autophagy, in this study we investigated the role of SYK in autophagy, MHC-IIexpression and adaptive immune response to OxLDL. We demonstrate that OxLDL induces autophagosome formation, MHC-IIexpression, and phosphorylation of SYK in macrophages. Gene knockout and pharmacological inhibitors of NOX2 and MAPK8 reduced OxLDL-induced autophagy. Using bone marrow-derived macrophages isolated from wild-type and myeloid-specific SYK knockout mice, we demonstrate that SYK regulates OxLDL-induced ROS generation, MAPK8 activation, BECN1-BCL2 dissociation, autophagosome formation and presentation of OxLDL-derived antigens to CD4+ T cells. ldlr−/− syk−/− mice fed a high-fat diet produced lower levels of IgG to malondialdehyde (MDA)-LDL, malondialdehyde-acetaldehyde (MAA)-LDL, and OxLDL compared to ldlr−/− mice. These results provide new insights into the mechanisms by which SYK regulates MHC-IIexpression via autophagy in macrophages and may contribute to regulation of adaptive immune responses in atherosclerosis. PMID:25946330

Monocyte class II major histocompatibility complex (MHC) expression is necessary for antigen presentation and stimulation of T-cells. The aim of this study was to correlate monocyte class IIMHC response to operative stress in children and the possible influence of cytokines in the postoperative period. We studied 21 children undergoing elective abdominal surgery. Operative stress score (OSS) was calculated. Monocyte class IIMHCexpression was measured preoperatively, immediately after surgery, 24 and 48 h postoperatively, using flow cytometry. Class IIMHC is expressed as mean fluorescence intensity (MFI) of monocytes expressingMHC (mean +/- SD). Cytokine levels (interleukins 1ra, 6, and 10, and tumor necrosis factor-alpha) were also measured. Data between time points were compared using repeated measures ANOVA. There was an immediate postoperative decrease in class IIMHCexpression, with lowest levels 24 h postoperatively (preoperative 50 +/- 23.6, 24 h 18.2 +/- 9.4, P < 0.0001 vs. preoperative). At 48 h there was partial recovery in class IIMHC, but levels were still significantly lower than preoperative (23.9 +/- 11.1, P < 0.001). The degree of monocyte depression was related to the magnitude of operative stress. Patients who had OSS <10 displayed some recovery in expression at 48 h 25.5 +/- 11.1), whereas in patients with OSS > or = 10 (severe surgical stress), expression further decreased at 48 h (MFI 14.0 +/- 0.1). There was an elevation of interleukin-1ra in the immediate postoperative period in both groups. There was no elevation in the other cytokines. Abdominal surgery in children decreases monocyte MHCexpression. Class IIMHC depression was related to magnitude of surgical trauma, implying that more severe immuneparesis follows surgery of greater magnitude. This may predispose to postoperative infection.

In adults, under physiological conditions proteins of the major histocompatibility complex, class II (MHCII) molecules are synthesized and then presented on the surface of the cells known under a common name as antigen presenting cells (APCs). Dendritic cells (DCs), microglia, macrophages, ameboid microglia and lymphocytes B are qualified as APCs. The aim of present study was to evaluate the expression of MHCII molecules in the central nervous system (CNS) and hematopoietic organs during the fetal development. Observations were made on the cerebral occipital lobe, cerebellum, thymus, spleen and liver of 30 normal human fetuses, between 11 and 22 week of gestation (GW). Histological, histochemical and immunohistochemical techniques were used to identify cells with expression of MHCII molecules. In the brain, MHCII molecules were detected on macrophages/ameboid microglia in meninges, choroid plexus and single cells of ramified microglia in deeper layers of the cortex and white matter. In the other organs besides macrophages and dendritic cells, MHCII molecules were also immunopositive in thymic epithelial cells, and in the spleen and liver also in other cells of stroma and lobule. The expression of MHCII molecules on so extensive population of cells, at an early stage of the fetal development, may evidence their significant involvement in histogenesis and morphogenesis. It seems that in adults the complex of MHCII with protein is originated from the foreign antigen. On the contrary, during normal fetal development the complex of MHCII with protein origins most probably from the fetus own structures.

Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class IIMHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class IIMHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class IIMHCexpressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class IIMHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways. PMID:9301537

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.

The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters. ?? 2006 Blackwell Munksgaard.

The major histocompatibility complex (MHC) is central to maintaining the immunologic vigor of individuals and populations. Classical MHC class II genes were targeted for partial sequencing in sea otters (Enhydra lutris) from populations in California, Washington, and Alaska. Sequences derived from sea otter peripheral blood leukocyte mRNAs were similar to those classified as DQA, DQB, DRA, and DRB in other species. Comparisons of the derived amino acid compositions supported the classification of these as functional molecules from at least one DQA, DQB, and DRA locus and at least two DRB loci. While limited in scope, phylogenetic analysis of the DRB peptide-binding region suggested the possible existence of distinct clades demarcated by geographic region. These preliminary findings support the need for additional MHC gene sequencing and expansion to a comprehensive study targeting additional otters.

MHC class II (MHC-II)-dependent antigen presentation by antigen-presenting cells (APCs) is carefully controlled to achieve specificity of immune responses; the regulated assembly and degradation of antigenic peptide-MHC-II complexes (pMHC-II) is one aspect of such control. In this study, we have examined the role of ubiquitination in regulating pMHC-II biosynthesis, endocytosis, recycling, and turnover in APCs. By using APCs obtained from MHC-II ubiquitination mutant mice, we find that whereas ubiquitination does not affect pMHC-II formation in dendritic cells (DCs), it does promote the subsequent degradation of newly synthesized pMHC-II. Acute activation of DCs or B cells terminates expression of the MHC-II E3 ubiquitin ligase March-I and prevents pMHC-II ubiquitination. Most importantly, this change results in very efficient pMHC-II recycling from the surface of DCs and B cells, thereby preventing targeting of internalized pMHC-II to lysosomes for degradation. Biochemical and functional assays confirmed that pMHC-II turnover is suppressed in MHC-II ubiquitin mutant DCs or by acute activation of wild-type DCs. These studies demonstrate that acute APC activation blocks the ubiquitin-dependent turnover of pMHC-II by promoting efficient pMHC-II recycling and preventing lysosomal targeting of internalized pMHC-II, thereby enhancing pMHC-II stability for efficient antigen presentation to CD4 T cells.

Genetic susceptibility to rheumatoid arthritis (RA) is associated with certain MHC class II molecules. To clarify the role of these determinants in RA, we generated the D1CC transgenic mouse that expressed genes involved in antigen processing and presentation by the MHC class II pathway in joints. The class II transactivator, which was transcribed from the rat collagen type II promoter and enhancer, directed the expression of these genes. In D1CC mice congenic for the H-2q (DBA/1) background, small amounts of bovine collagen type II in adjuvant induced reproducibly an inflammatory arthritis resembling RA. Importantly, these stimuli had no effect in DBA/1 mice. Eighty-nine percent of D1CC mice developed chronic disease with joint swelling, redness, and heat in association with synovial proliferation as well as pannus formation and mononuclear infiltration of synovial membranes. Granulomatous lesions resembling rheumatoid nodules and interstitial pneumonitis also were observed. As in patients with RA, anticyclic citrullinated peptide antibodies were detected during the inflammatory stage. Finally, joints in D1CC mice displayed juxtaarticular demineralization, severe joint space narrowing, and erosions, which led to ankylosis, but without the appearance of osteophytes. Thus, aberrant expression of MHC class II in joints facilitates the development of severe erosive inflammatory polyarthritis, which is very similar to RA. PMID:16980409

IL-10 is vastly studied for its anti-inflammatory properties on most immune cells. However, it has been reported that IL-10 activates B cells, up-regulates their MHC class II molecules and prevents apoptosis. As MARCH1 was shown to be responsible for the intracellular sequestration of MHC class II molecules in dendritic cells and monocytes in response to IL-10, we set out to clarify the role of this ubiquitin ligase in B cells. Here, we demonstrate in mice that splenic follicular B cells represent the major cell population that up-regulate MHCII molecules in the presence of IL-10. Activation of these cells through TLR4, CD40 or the IL-10 receptor caused the down-regulation of MARCH1 mRNA. Accordingly, B cells from MARCH1-deficient mice do not up-regulate I-A(b) in response to IL-10. In all, our results demonstrate that IL-10 can have opposite effects on MARCH1 regulation in different cell types.

Major histocompatibility complex class II (MHCII) expression is tightly regulated, being subjected to cell type-specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHCIIexpression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHCII in thymic epithelial cells (TECs). March8(-/-) mice have elevated MHCII at the surface of cortical TECs and autoimmune regulator (AIRE)(-) medullary TECs (mTECs), but not AIRE(+) mTECs. Despite this, thymic and splenic CD4(+) T cell numbers and repertoires remained unaltered in March8(-/-) mice. Notably, the ubiquitination of MHCII by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83(anu/anu) mice) have impaired CD4(+) T cell selection, but deleting March8 in Cd83(anu/anu) mice restored CD4(+) T cell selection to normal levels. Therefore, orchestrated regulation of MHCII surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4(+) T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation.

Major histocompatibility complex class II (MHCII) expression is tightly regulated, being subjected to cell type–specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHCIIexpression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHCII in thymic epithelial cells (TECs). March8−/− mice have elevated MHCII at the surface of cortical TECs and autoimmune regulator (AIRE)− medullary TECs (mTECs), but not AIRE+ mTECs. Despite this, thymic and splenic CD4+ T cell numbers and repertoires remained unaltered in March8−/− mice. Notably, the ubiquitination of MHCII by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83anu/anu mice) have impaired CD4+ T cell selection, but deleting March8 in Cd83anu/anu mice restored CD4+ T cell selection to normal levels. Therefore, orchestrated regulation of MHCII surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4+ T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation. PMID:27503069

MHCII-β chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHCII-β expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHCII-β expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHCII-β, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes.

The major histocompatibility complex (Mhc) is subject to pathogen-mediated balancing selection and can link natural selection with mate choice. We characterized two Mhc class II B loci in Leach's storm-petrel, Oceanodroma leucorhoa, focusing on exon 2 which encodes the portion of the protein that binds pathogen peptides. We amplified and sequenced exon 2 with locus-specific nested PCR and Illumina MiSeq using individually barcoded primers. Repeat genotyping of 78 single-locus genotypes produced identical results in 77 cases (98.7%). Sequencing of messenger RNA (mRNA) from three birds confirmed expression of both loci, consistent with the observed absence of stop codons or frameshifts in all alleles. In 48 birds, we found 9 and 12 alleles at the two loci, respectively, and all 21 alleles translated to unique amino acid sequences. Unlike many studies of duplicated Mhc genes, alleles of the two loci clustered into monophyletic groups. Consistent with this phylogenetic result, interlocus gene conversion appears to have affected only two short fragments of the exon. As predicted under a paradigm of pathogen-mediated selection, comparison of synonymous and non-synonymous substitution rates found evidence of a history of positive selection at putative peptide binding sites. Overall, the results suggest that the gene duplication event leading to these two loci is not recent and that point mutations and positive selection on the peptide binding sites may be the predominant forces acting on these genes. Characterization of these loci sets the stage for population-level work on the evolutionary ecology of Mhc in this species.

A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4(+) T cell responses are particularly desirable, as they enhance CD8(+) T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHCII on tumor cells, thereby promoting CD4(+) T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHCII and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHCIIexpression is likely due to the presence of invariant chain, in support of the MHCII(+)/Ii(-) paradigm.

Expression of major histocompatibility antigens class-2 (MHC-II) under non-inflammatory conditions is not usually associated with the nervous system. Comparative analysis of immunogenicity of human embryonic/fetal brain-derived neural stem cells (hNSCs) and human mesenchymal stem cells with neurogenic potential from umbilical cord (UC-MSCs) and paediatric adipose tissue (ADSCs), while highlighting differences in their immunogenicity, led us to discover subsets of neural cells co-expressing the neural marker SOX2 and MHC-II antigen in vivo during human CNS development. MHC-II proteins in hNSCs are functional, and differently regulated upon differentiation along different lineages. Mimicking an inflammatory response using the inflammatory cytokine IFNγ induced MHC-II up-regulation in both astrocytes and hNSCs, but not in UC-MSCs and ADSCs, either undifferentiated or differentiated, though IFNγ receptor expression was comparable. Together, hypoimmunogenicity of both UC-MSCs and ADSCs supports their suitability for allogeneic therapy, while significant immunogenicity of hNSCs and their progeny may at least in part underlie negative effects reported in some patients following embryonic neural cell grafts. Crucially, we show for the first time that MHC-IIexpression in developing human brains is not restricted to microglia as previously suggested, but is present in discrete subsets of neural progenitors and appears to be regulated independently of inflammatory stimuli. PMID:27080443

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription. In this article, we report that human NLRC5, which we recently showed to be involved in viral-mediated type I IFN responses, shuttles to the nucleus and activates MHC class I gene expression. Knockdown of NLRC5 in different human cell lines and primary dermal fibroblasts leads to reduced MHC class I expression, whereas introduction of NLRC5 into cell types with very low expression of MHC class I augments MHC class I expression to levels comparable to those found in lymphocytes. Expression of NLRC5 positively correlates with MHC class I expression in human tissues. Functionally, we show that both the N-terminal effector domain of NLRC5 and its C-terminal leucine-rich repeat domain are needed for activation of MHC class I expression. Moreover, nuclear shuttling and function depend on a functional Walker A motif. Finally, we identified a promoter sequence in the MHC class I promoter, the X1 box, to be involved in NLRC5-mediated MHC class I gene activation. Taken together, this suggested that NLRC5 acts in a manner similar to class II transactivator to drive MHCexpression and revealed NLRC5 as an important regulator of basal MHC class I expression.

The interaction of Fc receptors for IgG (FcγRs) on monocytes/macrophages with immune complexes (IC) triggers regulatory and effector functions. Previous studies have shown that FcγR–IC interactions inhibit the IFN-γ-induced expression of MHC class II in murine macrophages. However, the mechanism(s) responsible for these effects have not been elucidated. In addition, whether this IC-dependent effect also occurs in human cells is not known. Taking into account the fact that IC and IFN-γ are frequently found in infections and autoimmune disorders, together with the crucial role MHC class II molecules play in the regulation of immune response, we explored the effect and mechanism of IC-induced MHC class II down-regulation in human peripheral blood mononuclear cells (PBMC). This effect was studied either in the presence or absence of IFN-γ. We demonstrate that IC exert a drastic inhibition of basal and IFN-γ-induced expression of MHC class II on human monocytes. This effect was mediated through the interaction of IC with both FcγRI and FcγRII. Moreover, similar results were obtained using supernatants from IC-treated PBMC. The IC-induced down-regulation of MHC class II is abrogated by pepstatin and phosphoramidon, supporting the role of aspartic protease(s) and metalloprotease(s) in this process. In parallel with MHC class IIexpression, antigen presentation was markedly inhibited in the presence of IC. PMID:11529917

A failure of tight control of MHC class IIexpression on astrocytes may play a role in the development of autoimmune responses in multiple sclerosis. The 5-HT(4) serotonin receptor agonists cisapride and prucalopride, at concentrations between 10(-10) M and 10(-8) M, reduced interferon-gamma-induced MHC class II immunostaining in cultured astrocytes derived from newborn Wistar rats by approximately 50-60%. The magnitude of MHC class II inhibition by 5-HT(4) agonists was comparable to that of interferon-beta. The alpha(1)-adrenergic receptor agonist phenylephrine was without effect. Cisapride (10(-9) M) also prevented interferon-gamma-induced B7-1 and B7-2 immunostaining. Our results suggest that 5-HT(4) agonists may have therapeutic potential in multiple sclerosis by inhibiting the up-regulation of immune responsiveness of astrocytes in the central nervous system.

Genes of the major histocompatibility complex (MHC) are exceptionally polymorphic due to the combined effects of natural and sexual selection. Most research in wild populations has focused on the second exon of a single class II locus (DRB), but complete gene sequences can provide an illuminating backdrop for studies of intragenic selection, recombination, and organization. To this end, we characterized class II loci in the banner-tailed kangaroo rat (Dipodomys spectabilis). Seven DRB-like sequences (provisionally named MhcDisp-DRB*01 through *07) were isolated from spleen cDNA and most likely comprise > or =5 loci; this multiformity is quite unlike the situation in muroid rodents such as Mus, Rattus, and Peromyscus. In silico translation revealed the presence of important structural residues for glycosylation sites, salt bonds, and CD4+ T-cell recognition. Amino-acid distances varied widely among the seven sequences (2-34%). Nuclear DNA sequences from the Disp-DRB*07 locus (approximately 10 kb) revealed a conventional exon/intron structure as well as a number of microsatellites and short interspersed nuclear elements (B4, Alu, and IDL-Geo subfamilies). Rates of nucleotide substitution at Disp-DRB*07 are similar in both exons and introns (pi = 0.015 and 0.012, respectively), which suggests relaxed selection and may indicate that this locus is an expressed pseudogene. Finally, we performed BLASTn searches against Dipodomys ordii genomic sequences (unassembled reads) and find 90-97% nucleotide similarity between the two kangaroo rat species. Collectively, these data suggest that class II diversity in heteromyid rodents is based on polylocism and departs from the muroid architecture.

Stress responses have been associated with altered immunity and depending upon the type of stressor, can have diverse effects on disease outcomes. As the first line of defense against potential pathogens, alterations in cellular immune responses along the respiratory tract can have a significant impact on the manifestation of local and systemic disease. Utilizing a murine model of respiratory pneumonia, the current study investigated the effects of restraint stress on the induction of primary and secondary immunity along the respiratory tract, influencing host susceptibility. Female CD-1 mice were subjected to three hours of restraint stress over a period of four days followed by primary and secondary Streptococcus pneumoniae infection via intranasal route. Stress exposure led to increased retention of bacterial carriage in the lungs, enhanced polymorphonuclear cells and a preferential decrease in pulmonary CD11c+ MHCII+ cells resulting in delayed lethality during primary infection but significant impairment of acquired immune protection after secondary infection. We also provide evidence to support a role for lung-associated corticotrophin releasing hormone regulation through peripheral CRH and diverse CRH receptor expression by MHCII+ antigen presenting cells (APCs). We conclude that repeated restraint stress has distinct influences on immune cell populations that appear to be important in the generation of innate and adaptive immune responses along the respiratory tract with the potential to influence local and systemic protection against disease pathogenesis. PMID:18166336

Cattle DRA and DRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfected DRB gene clearly showed showed that it was DRB3 allele DRB3*0101, which corresponds to the 1D-IEF-determined allele DRBF3. 1D-IEF analysis of the tranfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigen-specific CD4{sup +} T cells from both the original animal used to obtain the genes, and also from an unrelated DRBF3{sup +} heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design. 45 refs., 5 figs., 1 tab.

To evaluate the impact of MHC antigen expression on the survival of patients with cancer, 77 human breast carcinomas were investigated for the expression of beta 2-microglobulin (beta 2m), HLA-A,B,C and HLA-DR. Thirty-one benign breast tumours were stained for comparison. The results for the carcinomas were related to the survival data of the cancer patients. The expression of beta 2m, HLA-A,B,C and HLA-DR was significantly lower in malignant tumours compared to the benign lesions. Whereas all benign tumours were positive for beta 2m and HLA-A,B,C and 28/31 positive for HLA-DR the following positivity rates were found in carcinomas: 74/77 for beta 2m, 57/77 for HLA-A,B,C and 10/77 for HLA-DR. The follow-up (median 45 months) of 66 cancer patients for overall survival and of 65 patients for disease-free survival revealed no influence of beta 2m, HLA-A,B,C or HLA-DR expression on the prognosis of this cancer. In conclusion, experimental data indicating the importance of MHC antigens in anti-tumour responses are not confirmed by the analysis of cancer patient survival data. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2201398

Genetic diversity estimation of the major histocompatibility complex (MHC) gene may be an important tool in the assessment of immune response ability against infectious disease. We were able to identify a near full-length expressed DQB sequence by RACE-PCR method from the Asiatic black bear, Ursus thibetanus in Japan. This is the first such full length expression in the Ursidae. The bear had at least one functional DQB locus. In phylogenetic tree analysis its DQB amino acid sequence formed a monophyletic group with DQB sequences from members of the order Carnivora and had a 90% nucleotide sequence similarity with the DQB allele of the California sea lion, Zalophus californianus. We compared the DQB amino acid composition of U. thibetanus with those of several other mammalian species including Homo sapiens. Amino acid residues known to be functionally important for human MHC genes, tended to be also conserved among other mammalian species while PBRs in the beta1 domain were heterogeneous among mammalian species. The DQB sequence obtained from the bear had not only no putative frameshifts or deletions but also no abnormal amino acid mutations such as had been observed in human DQB molecules. This suggests that the bear DQB sequence was an apparently functional DQB allele. As a preliminary study, we sequenced the exon 2 region of DQB alleles from genomic DNA, and succeeded to amplify the exon 2 of DQB loci. Our study will provide useful information for conservation genetics of the U. thibetanus as well as more generally regarding the mammalian MHC region.

Genetic risk for autoimmunity in HLA genes is most often attributed to structural specificity resulting in presentation of self-antigens. Autoimmune vitiligo is strongly associated with the MHC class II region. Here, we fine-map vitiligo MHC class II genetic risk to three SNPs only 47 bp apart, located within a predicted super-enhancer in an intergenic region between HLA-DRB1 and HLA-DQA1, localized by a genome-wide association study of 2,853 Caucasian vitiligo patients. The super-enhancer corresponds to an expression quantitative trait locus for expression of HLA-DR and HLA-DQ RNA; we observed elevated surface expression of HLA-DR (P = 0.008) and HLA-DQ (P = 0.02) on monocytes from healthy subjects homozygous for the high-risk SNP haplotype. Unexpectedly, pathogen-stimulated peripheral blood mononuclear cells from subjects homozygous for the high-risk super-enhancer haplotype exhibited greater increase in production of IFN-γ and IL-1β than cells from subjects homozygous for the low-risk haplotype. Specifically, production of IFN-γ on stimulation of dectin-1, mannose, and Toll-like receptors with Candida albicans and Staphylococcus epidermidis was 2.5- and 2.9-fold higher in high-risk subjects than in low-risk subjects, respectively (P = 0.007 and P = 0.01). Similarly, production of IL-1β was fivefold higher in high-risk subjects than in low-risk subjects (P = 0.02). Increased production of immunostimulatory cytokines in subjects carrying the high-risk haplotype may act as an “adjuvant” during the presentation of autoantigens, tying together genetic variation in the MHC with the development of autoimmunity. This study demonstrates that for risk of autoimmune vitiligo, expression level of HLA class II molecules is as or more important than antigen specificity. PMID:26787888

Major histocompatibility complex (MHC) class II deficiency is a primary immunodeficiency disease characterized by abnormality of MHC class II molecules surface expression on peripheral blood lymphocytes and monocytes. Clinical manifestations include extreme susceptibility to viral, bacterial, and fungal infections but the immunodeficiency is not as severe as SCID (severe combined immunodeficiency), as evidenced by failure to develop disseminated infection after BCG vaccination. Therefore, MHCII deficiency with BCGosis, that is disseminated BCGitis, is not reported commonly. We report an interesting case of BCGosis after vaccination that was diagnosed to have probable MHCII deficiency.

Major histocompatibility complex (MHC) class II molecules are expressed by antigen-presenting cells and stimulate CD4(+) T cells, which initiate humoral immune responses. Over the past decade, interest has developed to therapeutically impact the peptides to be exposed to CD4(+) T cells. Structurally diverse small molecules have been discovered that act on the endogenous peptide exchanger HLA-DM by different mechanisms. Exogenously delivered peptides are highly susceptible to proteolytic cleavage in vivo; however, it is only when successfully incorporated into stable MHCII-peptide complexes that these peptides can induce an immune response. Many of the small molecules so far discovered have highlighted the molecular interactions mediating the formation of MHCII-peptide complexes. As potential drugs, these small molecules open new therapeutic approaches to modulate MHCII antigen presentation pathways and influence the quality and specificity of immune responses. This review briefly introduces how CD4(+) T cells recognize antigen when displayed by MHC class II molecules, as well as MHC class II-peptide-loading pathways, structural basis of peptide binding and stabilization of the peptide-MHC complexes. We discuss the concept of MHC-loading enhancers, how they could modulate immune responses and how these molecules have been identified. Finally, we suggest mechanisms whereby MHC-loading enhancers could act upon MHC class II molecules.

Colorectal cancer is considered a non-immunogenic malignany. One strategy to augment the immunogenicity of such tumours is represented by the expression of costimulatory molecules by gene transfer. Using transfected variants of the human colorectal cancer cell line SW480 we tested various costimulatory molecules (CD80, CD86, CD54) and a class II major histocompatibility complex (MHC) allele (HLA-DR3) alone or in combination on their ability to support primary T-lymphocyte activation in vitro. Expression of CD80 or CD86 similarly as the combination of both was not sufficient to induce proliferation of human allogeneic T cells. Expression of CD54 together with CD80 strongly augmented the costimulatory function of CD80, as observed in the presence of a CD3 monoclonal antibody (mAb), but did not lead directly to a T-cell response against modified tumour cells. Importantly, SW480 cells coexpressing CD54, CD80 and the HLA-DR3 allele effectively promoted T-lymphocyte proliferation. Moreover, the use of such CD54+/CD80+/HLA-DR3+ SW480 variants for repetitive stimulations resulted in the generation of T-cell lines predominantly composed of CD8+ T cells exhibiting class I MHC restricted cytolytic activity towards untransfected SW480 tumour cells. This demonstrates that the generation of immunogenic tumour cell variants, i.e. for the use as cellular vaccines, requires multiple genetic alterations in the case of non-immunogenic human tumours cells, such as colorectal cancer cells.

MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.

Bone marrow-derived mast cells as well as dendritic cells, macrophages and B lymphocytes express major histocompatibility complex (MHC) class II molecules. In mast cells, the majority of MHC class II molecules reside in intracellular cell type-specific compartments, secretory granules. To understand the molecular basis for the localisation of MHC class II molecules in secretory granules, MHC class II molecules were expressed, together with the invariant chain, in the mast cell line, RBL-2H3. Using electron and confocal microscopy, we observed that in RBL-2H3 cells, mature and immature class II molecules accumulate in secretory granules. Two particular features of class II transport accounted for this intracellular localization: first, a large fraction of newly synthesized MHC class II molecules remained associated with invariant chain fragments. This defect, resulting in a slower rate of MHC class II maturation, was ascribed to a low cathepsin S activity. Second, although a small fraction of class II dimers matured (i.e. became free of invariant chain), allowing their association with antigenic peptides, they were retained in secretory granules. As a consequence of this intracellular localization, cell surface expression of class II molecules was strongly increased by cell activation stimuli which induced the release of the contents of secretory granules. Our results suggest that antigen presentation, and thereby antigen specific T cell stimulation, are regulated in mast cells by stimuli which induce mast cell activation.

The study of T cell positive selection in the thymus has long been focused on the specificity of the MHC-TCR interactions, making use of genetically manipulated mice that display TCR specificities or selecting peptides of limited diversity. However, little is known on the role of the MHC molecules irrespective of the peptide specificity and the implications of MHC multigenic structure in thymic positive selection have not been addressed. Here, we investigated the effect of MHC class II genetic configuration on the positive selection efficiency of naturally generated pre-selection repertoires in the mouse thymus. Analysis of positively selected thymocyte populations in MHC-congenic and -transgenic mice revealed that expression of I-E molecule in the thymic cortex increases positive selection efficiency of CD4 cells by approximately 50%. We show that increments in positive selection attributable to either the I-A and I-E genes are not due to increased MHC class IIexpression in the thymic cortex and are not affected by the number of MHC alleles. Collectively, our findings imply that MHC class II gene-restricted TCR specificities significantly contribute to positive selection efficiency, introducing the notion that multigenic structure of the MHC locus serves to increase selection of non-overlapping TCR repertoires.

Antigen presentation by major histocompatibility complex (MHC) proteins is essential for adaptive immunity. Prior to presentation, peptides need to be generated from proteins that are either produced by the cell’s own translational machinery or that are funneled into the endo-lysosomal vesicular system. The prolonged interaction between a T cell receptor and specific pMHC complexes, after an extensive search process in secondary lymphatic organs, eventually triggers T cells to proliferate and to mount a specific cellular immune response. Once processed, the peptide repertoire presented by MHC proteins largely depends on structural features of the binding groove of each particular MHC allelic variant. Additionally, two peptide editors—tapasin for class I and HLA-DM for class II—contribute to the shaping of the presented peptidome by favoring the binding of high-affinity antigens. Although there is a vast amount of biochemical and structural information, the mechanism of the catalyzed peptide exchange for MHC class I and class II proteins still remains controversial, and it is not well understood why certain MHC allelic variants are more susceptible to peptide editing than others. Recent studies predict a high impact of protein intermediate states on MHC allele-specific peptide presentation, which implies a profound influence of MHC dynamics on the phenomenon of immunodominance and the development of autoimmune diseases. Here, we review the recent literature that describe MHC class I and II dynamics from a theoretical and experimental point of view and we highlight the similarities between MHC class I and class II dynamics despite the distinct functions they fulfill in adaptive immunity. PMID:28367149

The polymorphism of major histocompatibility complex (MHC) class II DQ and DR genes in five common equine leukocyte antigen (ELA) haplotypes was determined through sequencing of mRNA transcripts isolated from lymphocytes of eight ELA homozygous horses. Ten expressedMHC class II genes were detected in horses of the ELA-A3 haplotype carried by the donor horses of the equine bacterial artificial chromosome (BAC) library and the reference genome sequence: four DR genes and six DQ genes. The other four ELA haplotypes contained at least eight expressed polymorphic MHC class II loci. Next generation sequencing (NGS) of genomic DNA of these four MHC haplotypes revealed stop codons in the DQA3 gene in the ELA-A2, ELA-A5, and ELA-A9 haplotypes. Few NGS reads were obtained for the other MHC class II genes that were not amplified in these horses. The amino acid sequences across haplotypes contained locus-specific residues, and the locus clusters produced by phylogenetic analysis were well supported. The MHC class II alleles within the five tested haplotypes were largely non-overlapping between haplotypes. The complement of equine MHC class II DQ and DR genes appears to be well conserved between haplotypes, in contrast to the recently described variation in class I gene loci between equine MHC haplotypes. The identification of allelic series of equine MHC class II loci will aid comparative studies of mammalian MHC conservation and evolution and may also help to interpret associations between the equine MHC class II region and diseases of the horse.

CD4+ T cells have been shown to reject tumor cells with no detectable expression of major histocompatibility complex class II (MHCII). However, under certain circumstances, induction of ectopic MHCIIexpression on tumor cells has been reported. To confirm that CD4+ T cell-mediated anti-tumor immunity can be successful in the complete absence of antigen display on the tumor cells themselves, we eliminated MHCII on tumor cells using CRISPR/Cas9. Our results demonstrate that ablation of the relevant MHCII (I-Ed) in multiple myeloma cells (MOPC315) does not hinder rejection by tumor-specific CD4+ T cells. These findings provide conclusive evidence that CD4+ T cells specific for tumor antigens can eliminate malignant cells in the absence of endogenous MHC class IIexpression on the tumor cells. This occurs through antigen uptake and indirect presentation on tumor-infiltrating macrophages. PMID:27626487

The aim was to investigate the distribution of CD2(+), CD4(+) and CD8(+) lymphocyte subpopulations and MHC class IIexpressing cells in the sow endometrium following pre-ovulatory insemination and during early pregnancy. Crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were inseminated once at 15-20 h before ovulation. The sows were slaughtered at 5-6h (group I, n=4) after AI or at 20-25 h (group II, n=4) and 70 h (group III, n=4) after ovulation, day 11 (group IV, day 1=first day of standing oestrus, n=3) and day 19 (group V, n=3). Uterine horns were flushed to control for the presence of spermatozoa and neutrophils (groups I-IV) and/or for recovery of oocytes and/or embryos (groups II-IV, control of pregnancy). Cryofixed mesometrial uterine samples were analysed by immunohistochemistry with an avidin-biotin-peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface (SE) and glandular (GE) epithelia as well as connective tissue layers in subepithelial (SL) and glandular (GL) areas were examined by light microscopy. Taking all groups and different tissue layers together, the most commonly observed positive cells were CD2(+) cells (P=0.001). The largest number of CD2(+) cells within the SE was observed in groups I and II, and the smallest number in group V. In the SE and GE, more CD8(+) (T cytotoxic) cells were observed than CD4(+) (T helper) cells. In the SL and GL, the largest numbers of CD2(+), CD4(+) and CD8(+) cells were found in group V. Taking all groups together, a larger number of CD4(+) cells compared with CD8(+) cells were found. For the proportion of (CD4(+)+CD8(+))/CD2(+) cells, there were significantly (P=0.001) lower proportions in the SE and GE than in the SL and GL. A larger number of MHC class IIexpressing cells in the SE was observed in groups I, II and III compared with the other groups. In the SL, a larger number of MHC class IIexpressing cells was observed in groups

Kupffer cells (KC), by virtue of their ability to present antigen (AP) and express major histocompatibility complex (MHC) class II antigen (Ia), play a pivotal role in the host defence system against invading micro-organisms. Although haemorrhagic shock depresses the above KC functions, it is not known whether increased KC tumour necrosis factor (TNF) production and elevated TNF plasma levels following haemorrhage are responsible for it. To study this, C3H/HeN mice were pretreated intraperitoneally with either anti-murine TNF antibody (anti-TNF Ab) or saline. Twenty hours later mice were bled and maintained at a mean blood pressure of 35 mmHg for 60 min followed by adequate fluid resuscitation. Two and 24 hr later, plasma was collected and KC were isolated. AP was measured by co-culturing KC with the D10.G4.1 Th cell clone. Ia expression was determined by direct immunofluorescence. Interleukin (IL)-1, IL-6 and TNF levels in KC supernatants and plasma were measured with bioassays or ELISA. Haemorrhage increased circulating TNF levels by 215% at 2 hr and by 76% at 24 hr (P less than 0.05), which was prevented by pretreatment with anti-TNF Ab. Haemorrhage-induced increase of circulating IL-6 was abolished (P less than 0.05) at 2 hr but not at 24 hr in the anti-TNF Ab group. The suppression of KC AP (P less than 0.05) and Ia expression (P less than 0.05) due to haemorrhage was attenuated (P less than 0.05) in anti-TNF Ab-treated mice at 2 and 24 hr and KC IL-1 and TNF synthesis was further (P less than 0.01) increased. These results indicate that TNF plays a critical role in the initiation and regulation of KC AP, Ia expression, and cytokine production following haemorrhage. PMID:1748476

Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHCIIexpression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHCIIexpression and presentation by inhibiting the transcription of MHCII transactivator (CIITA) promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHCII molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters). This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHCII presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.

Major histocompatibility complex (MHC) class II molecules present exogenously derived antigen peptides to CD4 T cells, driving activation of naïve T cells and supporting CD4-driven immune functions. However, MHC class II molecules are not inert protein pedestals that simply bind and present peptides. These molecules also serve as multi-functional signaling molecules delivering activation, differentiation, or death signals (or a combination of these) to B cells, macrophages, as well as MHC class II-expressing T cells and tumor cells. Although multiple proteins are known to associate with MHC class II, interaction with STING (stimulator of interferon genes) and CD79 is essential for signaling. In addition, alternative transmembrane domain pairing between class II α and β chains influences association with membrane lipid sub-domains, impacting both signaling and antigen presentation. In contrast to the membrane-distal region of the class II molecule responsible for peptide binding and T-cell receptor engagement, the membrane-proximal region (composed of the connecting peptide, transmembrane domain, and cytoplasmic tail) mediates these “non-traditional” class II functions. Here, we review the literature on the function of the membrane-proximal region of the MHC class II molecule and discuss the impact of this aspect of class II immunobiology on immune regulation and human disease. PMID:27006762

The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.

Background Computational methods for the prediction of Major Histocompatibility Complex (MHC) class II binding peptides play an important role in facilitating the understanding of immune recognition and the process of epitope discovery. To develop an effective computational method, we need to consider two important characteristics of the problem: (1) the length of binding peptides is highly flexible; and (2) MHC molecules are extremely polymorphic and for the vast majority of them there are no sufficient training data. Methods We develop a novel string kernel MHC2SK (MHC-II String Kernel) method to measure the similarities among peptides with variable lengths. By considering the distinct features of MHC-II peptide binding prediction problem, MHC2SK differs significantly from the recently developed kernel based method, GS (Generic String) kernel, in the way of computing similarities. Furthermore, we extend MHC2SK to MHC2SKpan for pan-specific MHC-II peptide binding prediction by leveraging the binding data of various MHC molecules. Results MHC2SK outperformed GS in allele specific prediction using a benchmark dataset, which demonstrates the effectiveness of MHC2SK. Furthermore, we evaluated the performance of MHC2SKpan using various benckmark data sets from several different perspectives: Leave-one-allele-out (LOO), 5-fold cross validation as well as independent data testing. MHC2SKpan has achieved comparable performance with NetMHCIIpan-2.0 and outperformed NetMHCIIpan-1.0, TEPITOPEpan and MultiRTA, being statistically significant. MHC2SKpan can be freely accessed at http://datamining-iip.fudan.edu.cn/service/MHC2SKpan/index.html. PMID:24564280

The class II transactivator (CIITA) regulates expression of the classical and non-classical MHC class II genes, HLA-DR, -DP, -DQ and -DM, but not the B cell-specific HLA-DO (DO). Here we show that only HLA-DR expression is completely dependent on CIITA, since residual expression of HLA-DM, -DP and the beta chain of DQ was observed in CIITA-deficient RJ2.2.5 cells. Although DO shows a unique expression pattern compared to other MHC class II genes, prolonged IFN-gamma treatment of HeLa cells induced DOB expression. Similar to all MHC class II promoters, the DOB promoter contains the highly conserved W, X1, and Y boxes in addition to a putative OCT box. Mutational analysis of the DOB promoter demonstrated that the X1, Y and OCT boxes are necessary for maximum promoter activity.Furthermore, our results demonstrate that CREB-1, RFXANK and Oct-2 occupy the DOB promoter in vivo, However, CIITA and Bob-1 were only minimally recruited. Finally, fusion of Bjab, a DOB-negative B cell line, with.174 B cells that lack the complete MHC class II region (including the DO genes), lead to DO expression. These data indicate that the expression of DO is regulated by an unidentified factor in B cells.

MHC class II and CD1-mediated antigen presentation on various APCs [B cells, monocytes, and dendritic cells (DC)] are subject to at least three distinct levels of regulation. The first one concerns the expression and structure of the antigen-presenting molecules; the second is based on the extracellular environment and signals of danger detected. However, a third level of regulation, which has been largely overlooked, is determined by lateral associations between antigen-presenting molecules and other proteins, their localization in specialized microdomains within the plasma membrane, and their trafficking pathways. This review focuses on features common to MHCII and CD1 molecules in their ability to activate specific T lymphocytes with the objective of addressing one basic question: What are the mechanisms regulating antigen presentation by MHCII and CD1 molecules within the same cell? Recent studies in immature DC, where MHCII and CD1 are coexpressed, suggest that the invariant chain (Ii) regulates antigen presentation by either protein. Ii could therefore favor MHCII or CD1 antigen presentation and thereby discriminate between antigens.

The tremendous progress in the field of basic immunology and immunochemistry made in the last decade has significantly advanced our understanding of antigen processing and presentation by MHC class I and II proteins. In this review different techniques to study peptide interaction with MHC class II molecules are summarized and their impact on the elucidation of quantitative parameters, like affinities or kinetic data, is discussed. A recently introduced method based on synthetic combinatorial peptide libraries allows to quantify the binding contribution of each amino acid residue in a class II ligand and is presented in more detail. As this knowledge is fundamental for current investigations in modern medicine, e.g. for novel immune system based therapy concepts, further aspects like the design of new high affinity MHC class II ligands and the prediction of peptide antigens are discussed.

This study aimed to analyse the expression of myosin heavy chain (MHC) isoforms in bovine muscles, with particular attention to the MHC-2B gene. Diaphragm, longissimus dorsi, masseter, several laryngeal muscles and two extraocular muscles (rectus lateralis and retractor bulbi) were sampled in adult male Bos taurus (age 18-24 months, mass 400-500 kg) and analysed by RT-PCR, gel electrophoresis and immunohistochemistry. Transcripts and proteins corresponding to eight MHC isoforms were identified: MHC-alpha and MHC-beta/slow (or MHC-1), two developmental isoforms (MHC-embryonic and MHC-neonatal), three adult fast isoforms (MHC-2A, MHC-2X and MHC-2B) and the extraocular isoform MHC-Eo. All eight MHC isoforms were found to be co-expressed in extrinsic eye muscles, retractor bulbi and rectus lateralis, four (beta/slow, 2A, 2X, neonatal) in laryngeal muscles, three (beta/slow, 2A and 2X) in trunk and limb muscles and two (beta/slow and alpha) in masseter. The expression of MHC-2B and MHC-Eo was restricted to extraocular muscles. Developmental MHC isoforms (neonatal and embryonic) were only found in specialized muscles in the larynx and in the eye. MHC-alpha was only found in extraocular and masseter muscle. Single fibres dissected from masseter, diaphragm and longissimus were classified into five groups (expressing, respectively, beta/slow, alpha, slow and 2A, 2A and 2X) on the basis of MHC isoform electrophoretical separation, and their contractile properties [maximum shortening velocity (v(0)) and isometric tension (P(0))] were determined. v(0) increased progressively from slow to fast 2A and fast 2X, whereas hybrid 1-2A fibres and fibres containing MHC-alpha were intermediate between slow and fast 2A.

Major histocompatibility complex (MHC) genes are important for vertebrate immune response and typically display high levels of diversity due to balancing selection from exposure to diverse pathogens. An understanding of the structure of the MHC region and diversity among functional MHC genes is critical to understanding the evolution of the MHC and species resilience to disease exposure. In this study, we characterise the structure and diversity of class IIMHC genes in little spotted kiwi Apteryx owenii, a ratite bird representing the basal avian lineage (paleognaths). Results indicate that little spotted kiwi have a more complex MHC structure than that of other non-passerine birds, with at least five class IIMHC genes, three of which are expressed and likely to be functional. Levels of MHC variation among little spotted kiwi are extremely low, with 13 birds assayed having nearly identical MHC genotypes (only two genotypes containing four alleles, three of which are fixed). These results suggest that recent genetic drift due to a species-wide bottleneck of at most seven birds has overwhelmed past selection for high MHC diversity in little spotted kiwi, potentially leaving the species highly susceptible to disease.

The human MHC class II DNA gene was identified and sequenced by Trowsdale and Kelly. When a molecular map of the HLA-D region became available, it was shown that the HLA-DNA gene was unusual in not having a B gene partner situated within a few kilobases (kb), the nearest B gene being HLA-DPB1. The nearest unpaired B gene is HLA-DOB which is approximately 160 kb telomeric of HLA-DNA. More recently, the mouse MHC class II genes H-20A and H-20B were shown to be equivalent to the HLA-DNA and HLA-DOB genes. Moreover, the mouse genes expressed an MHC class II protein whose tissue distribution was restricted to B cells and epithelial cell of the thymic medulla. No corresponding HLA-DN protein has been reported. 21 refs., 3 figs.

The success of immunotherapy relies on the participation of all arms of the immune system and the role of CD4+ T lymphocytes in preventing tumor growth is now well established. Understanding how tumors evade immune responses holds the key to the development of cancer immunotherapies. In this review, we discuss how MHC Class IIexpression varies in cancer cells and how this influences antitumor immune responses. We also discuss the means that are currently available for harnessing the MHC Class II antigen presentation pathway for the development of efficient vaccines to activate the immune system against cancer.

Ecological conditions can influence not only the expression of a phenotype, but also the heritability of a trait. As such, heritable variation for a trait needs to be studied across environments. We have investigated how pathogen challenge affects the expression of MHC genes in embryos of the lake whitefish Coregonus palaea. In order to experimentally separate paternal (i.e. genetic) from maternal and environmental effects, and determine whether and how stress affects the heritable variation for MHCexpression, embryos were produced in full-factorial in vitro fertilizations, reared singly, and exposed at 208 degree days (late-eyed stage) to either one of two strains of Pseudomonas fluorescens that differ in their virulence characteristics (one increased mortality, while both delayed hatching time). Gene expression was assessed 48 h postinoculation, and virulence effects of the bacterial infection were monitored until hatching. We found no evidence of MHC class IIexpression at this stage of development. MHC class I expression was markedly down-regulated in reaction to both pseudomonads. While MHCexpression could not be linked to embryo survival, the less the gene was expressed, the earlier the embryos hatched within each treatment group, possibly due to trade-offs between immune function and developmental rate or further factors that affect both hatching timing and MHCexpression. We found significant additive genetic variance for MHC class I expression in some treatments. That is, changes in pathogen pressures could induce rapid evolution in MHC class I expression. However, we found no additive genetic variance in reaction norms in our study population.

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressedMHC class I loci and two expressedMHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.

Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment-admittedly with limited geographic coverage and sample size-did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.

During the last decade, the major histocompatibility complex (MHC) has received much attention in the fields of evolutionary and conservation biology because of its potential implications in many biological processes. New insights into the gene structure and evolution of MHC genes can be gained through study of additional lineages of birds not yet investigated at the genomic level. In this study, we characterized MHC class II B genes in five families of birds of prey (Accipitridae, Pandionidae, Strigidae, Tytonidae, and Falconidae). Using PCR approaches, we isolated genomic MHC sequences up to 1300 bp spanning exons 1 to 3 in 26 representatives of each raptor lineage, finding no stop codons or frameshift mutations in any coding region. A survey of diversity across the entirety of exon 2 in the lesser kestrel Falco naumanni reported 26 alleles in 21 individuals. Bayesian analysis revealed 21 positively selected amino acid sites, which suggests that the MHC genes described here are functional and probably expressed. Finally, through interlocus comparisons and phylogenetic analysis, we also discuss genetic evidence for concerted and transspecies evolution in the raptor MHC.

Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34+CD117+CD41+CD151+ megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressedMHC Class II in contrast to platelets and functioned as professional APC before they matured further. Moreover, MKp constitutively expressed mRNA encoding mediators for human Th17 expansion, including IL-1, IL-18, IL-6, TGFβ, IL-23, BAFF, and COX2. MKp also expressed high levels of type I interferon and IRF5 mRNA. In contrast to platelets, MKp augmented the expansion of Th17, Th1, and potent Th17/Th1 double-positive cells in normal PBMC and CD4 line T cells from normal subjects or lupus patients. The Th cell augmentation involved pre-committed memory cells, and was significant although modest, because only non-cognate MKp-T cell interactions could be studied, under non-polarizing conditions. Importantly, the MKp-mediated expansion was observed in the presence or absence of direct MKp-T cell contact. Furthermore, MKp augmented Th17 responses against Candida albicans, a serious opportunistic pathogen. These results indicate an immunologic role of MKp in situations associated with extramedullary hematopoiesis and mobilization of HSPC. PMID:25454068

pRB is required for IFN-gamma-induction of MHC class II in human tumor cell lines, providing a potential link between tumor suppressors and the immune system. However, other genes, such as cyclin D1, show pRB-dependency only in tumor cells, so by analogy, pRB may not be necessary for cII-regulation in normal cells. Here, we demonstrate that induction of the mouse MHC class II I-A heterodimer is normal in RB+/+ mouse embryonic fibroblasts (MEFs), but deficient in RB-/- MEFs. Inducibility is restored in RB-/- MEFs stably transfected with wild type RB cDNA or infected with an adenovirus expressing pRB. Thus, involvement of pRB in MHC class IIexpression is conserved in the mouse and is not an aberrant feature of tumorigenic, aneuploid, human tumor cells. Although cII genes are generally induced in a coordinate fashion, suggesting a common mechanism, we found that pRB was specifically required for induction of the Abeta, but not Aalpha or other MHC cII genes including Ebeta, Ii and H2-Malpha. Finally, IFN-gamma-induction of class II transactivator (CIITA), was pRB-independent, suggesting that pRB works downstream of this master-regulator of MHC class IIexpression.

MHCII, a major feature of the adaptive immune system, is lacking in Atlantic cod, and there are different scenarios (metabolic cost hypothesis or functional shift hypothesis) that might explain this loss. The lack of MHCII coincides with an increased number of genes for MHC I and Toll-like receptors (TLRs).

Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4(+) T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection.

Background Cordyceps militarys water extract (CME) has been reported to exert antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not yet been elucidated. In this study, we examined the effects of CME on the antigen presenting function of antigen presenting cells (APCs). Methods Dendritic cells (DCs) were cultured in the presence of CME, and then allowed to phagocytose microspheres containing ovalbumin (OVA). After washing and fixing the efficacy of OVA, peptide presentation by DCs were evaluated using CD8 and CD4 T cells. Also, we confirmed the protein levels of proinflammatory cytokines through western blot analysis. Results CME enhanced both MHC class I and class II-restricted presentation of OVA in DCs. In addition, the expression of both MHC class I and II molecules was enhanced, but there was no changes in the phagocytic activity of exogenous OVA. Furthermore, CME induced the protein levels of iNOS, COX-2, proinflammatory cytokines, and nuclear p65 in a concentration-dependent manner, as determined by western blot. Conclusion These results provide an understanding of the mechanism of the immuno-enhancing activity of CME on the induction of MHC-restricted antigen presentation in relation to their actions on APCs. PMID:20844738

Macrophages are critically involved in the interaction between T. crassiceps and the murine host immune system. Also, a strong gender-associated susceptibility to murine cysticercosis has been reported. Here, we examined the sex-associated expression of molecules MHC-II, CD80, CD86, PD-L1, and PD-L2 on peritoneal F4/80(hi) macrophages of BALB/c mice infected with Taenia crassiceps. Peritoneal macrophages from both sexes of mice were exposed to T. crassiceps total extract (TcEx). BALB/c Females mice recruit higher number of macrophages to the peritoneum. Macrophages from infected animals show increased expression of PDL2 and CD80 that was dependent from the sex of the host. These findings suggest that macrophage recruitment at early time points during T. crassiceps infection is a possible mechanism that underlies the differential sex-associated susceptibility displayed by the mouse gender.

Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressedMHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.

Major histocompatibility complex (MHC) is a multi-gene family that is very suitable to investigate a wide range of open questions in evolutionary ecology. In this study, we characterized two expressedMHC class II B genes (DAB1 and DAB2) in the Grey Heron (Aves: Ardea cinerea). We further developed the primer pairs to amplify and sequence two MHC class II B loci in ten ardeid birds. Phylogenetic analysis revealed that different parts of the genes showed different evolutionary patterns. The exon 2 sequences tended to cluster two gene-specific lineages. In each lineage, exon 2 sequences from several species showed closer relationships than sequences within species, and two shared identical alleles were found between species (Egretta sacra and Nycticorax nycticorax; Egretta garzetta and Bubulcus ibis), supporting the hypothesis of trans-species polymorphism. In contrast, the species-specific intron 2 plus partial exon 3 tree suggested that DAB1 and DAB2 were subject to concerted evolution. GENECONV analyses showed the gene exchange played an important role in the ardeid MHC evolution.

Major histocompatibility complex class II (MHCII) molecules are expressed on the surface of antigen presenting cells and display short bound peptide fragments derived from self and nonself antigens. These peptide-MHC complexes function to maintain immunological tolerance in the case of self antigens and initiate the CD4+ T cell response in the case of foreign proteins. Here we report the application of LC-MS/MS analysis to identify MHCII peptides derived from endogenous proteins expressed in freshly isolated murine splenic DCs. The cell number was enriched in vivo upon treatment with Flt3L-B16 melanoma cells. In a typical experiment, starting with about 5× 108 splenic DCs, we were able to reliably identify a repertoire of over 100 MHCII peptides originating from about 55 proteins localized in membrane (23%), intracellular (26%), endo-lysosomal (12%), nuclear (14%) and extracellular (25%) compartments. Using synthetic isotopically labeled peptides corresponding to the sequences of representative bound MHCII peptides, we quantified by LC-MS relative peptide abundance. In a single experiment, peptides were detected in a wide concentration range spanning from 2.5 fmol/μL to 12 pmol/μL or from approximately 13 copies to 2×105 copies per DC. These peptides were found in similar amounts on B cells where we detected about 80 peptides originating from 55 proteins distributed homogenously within the same cellular compartments as in DCs. About 90 different binding motifs predicted by the epitope prediction algorithm were found within the sequences of the identified MHCII peptides. These results set a foundation for future studies to quantitatively investigate the MHCII repertoire on DCs generated under different immunization conditions. PMID:21913724

Peptide loading of major histocompatibility complex (MHC) class II molecules in the endosomes and lysosomes of antigen-presenting cells is catalyzed by human leukocyte antigen-DM (HLA-DM) and modulated by HLA-DO. In a structural study in this issue, Guce et al. show that HLA-DO is an MHC class II mimic and functions as a competitive and essentially irreversible inhibitor of HLA-DM activity, thereby inhibiting MHC class II antigen presentation.

The major histocompatibility complex (MHC) plays a crucial role in the immune system, and in some species, it is a target by which individuals choose mates to optimize the fitness of their offspring, potentially mediated by olfactory cues. Under the genetic compatibility hypothesis, individuals are predicted to choose mates with compatible MHC alleles, to increase the fitness of their offspring. Studies of MHC-based mate choice in wild mammals are under-represented currently, and few investigate more than one class of MHC genes. We investigated mate choice based on the compatibility of MHC class I and II genes in a wild population of European badgers (Meles meles). We also investigated mate choice based on microsatellite-derived pairwise relatedness, to attempt to distinguish MHC-specific effects from genomewide effects. We found MHC-assortative mating, based on MHC class II, but not class I genes. Parent pairs had smaller MHC class II DRB amino acid distances and smaller functional distances than expected from random pairings. When we separated the analyses into within-group and neighbouring-group parent pairs, only neighbouring-group pairs showed MHC-assortative mating, due to similarity at MHC class II loci. Our randomizations showed no evidence of genomewide-based inbreeding, based on 35 microsatellite loci; MHC class II similarity was therefore the apparent target of mate choice. We propose that MHC-assortative mate choice may be a local adaptation to endemic pathogens, and this assortative mate choice may have contributed to the low MHC genetic diversity in this population.

In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHCII surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHCII KO clones. Peptide loaded B16F10 MHCII KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor. PMID:28301575

Among other features, peptides affect MHC class II molecules, causing changes in the binding of bacterial superantigens (b-Sag). Whether peptides can alter binding of viral superantigens (v-Sag) to MHC class II was not known. Here we addressed the question of whether mutations limiting the diversity of peptides bound by the MHC class II molecules influenced the presentation of v-Sag and, subsequently, the life cycle of the mouse mammary tumor virus (MMTV). T cells reactive to v-Sag were found in mice lacking DM molecules as well as in A(b)Ep-transgenic mice in which MHC class II binding grooves were predominantly occupied by an invariant chain fragment or Ealpha(52-68) peptide, respectively. APCs from the mutant mice failed to present v-Sag, as determined by the lack of Sag-specific T cell activation, Sag-induced T cell deletion, and by the aborted MMTV infection. In contrast, mice that express I-A(b) with a variety of bound peptides presented v-Sag and were susceptible to MMTV infection. Comparison of v-Sag and b-Sag presentation by the same mutant cells suggested that presentation of v-Sag had requirements similar to that for presentation of toxic shock syndrome toxin-1. Thus, MHC class II peptide repertoire is critical for recognition of v-Sag by the T cells and affects the outcome of infection with a retrovirus.

HLA-DM is a major histocompatibility complex (MHC) class II-like molecule that facilitates antigen processing by catalyzing the exchange of invariant chain-derived peptides (CLIP) from class II molecules for antigenic peptides. HLA-DO is a second class II-like molecule that physically associates with HLA-DM in B cells. HLA-DO was shown to block HLA-DM function. Purified HLA-DM-DO complexes could not promote peptide exchange in vitro. Expression of HLA-DO in a class II+ and DM+, DO- human T cell line caused the accumulation of class II-CLIP complexes, indicating that HLA-DO blocked DM function in vivo and suggesting that HLA-DO is an important modulator of class II-restricted antigen processing.

Misfolded proteins localized in the endoplasmic reticulum are degraded promptly and thus are not transported outside cells. However, misfolded proteins in the endoplasmic reticulum are rescued from protein degradation upon association with major histocompatibility complex (MHC) class II molecules and are transported to the cell surface by MHC class II molecules without being processed to peptides. Studies on the misfolded proteins rescued by MHC class II molecules have revealed that misfolded proteins associated with MHC class II molecules are specific targets for autoantibodies produced in autoimmune diseases. Furthermore, a strong correlation has been observed between autoantibody binding to misfolded proteins associated with MHC class II molecules and the autoimmune disease susceptibility conferred by each MHC class II allele. These new insights into MHC class II molecules suggest that misfolded proteins rescued from protein degradation by MHC class II molecules are recognized as "neo-self" antigens by immune system and are involved in autoimmune diseases as autoantibody targets.

Background Inflammatory bowel disease (IBD) pathogenesis involves an inadequately controlled immune reaction to intestinal microbiota and CD4+ T cells, dependent upon MHC class II (MHC-II) processing and presentation by antigen presenting cells (APC), play important roles The role of professional APC (macrophages and dendritic cells (DC)) and nonprofessional APC (intestinal epithelial cells (IEC)) in microbial driven intestinal inflammation remains controversial. Methods We generated transgenic animals on a MHC-II−/− genetic background in which MHC-II is expressed on a) DC via the CD11c promoter (CD11cTg) or b) IEC via the fatty acid binding protein (liver) promoter (EpithTg). These mice were crossed with Rag2−/− mice to eliminate T and B cells (CD11cTg/Rag2−/− and EpithTg/Rag2−/−). Helicobacter bilis (Hb) infection and adoptive transfer (AT) of naïve CD4+ T cells were used to trigger IBD. Results CD11cTg/Rag2−/− mice infected with Hb+AT developed severe colitis within three weeks post AT, similar to disease in positive control Rag2−/− mice infected with Hb+AT. CD11cTg/Rag2−/− mice given AT alone or Hb alone had significantly less severe colitis. In contrast, EpithTg/Rag2−/− mice infected with Hb+AT developed mild colitis by three weeks and even after 16 weeks post adoptive transfer, had only mild lesions. Conclusions MHC-IIexpression restricted to DCs is sufficient to induce severe colitis in the presence of T cells and a microorganism such as Hb within three weeks of adoptive transfer. Expression of MHC-II solely on IEC in the presence of a microbial trigger and T cells was insufficient for triggering severe colitis. PMID:22619032

Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator), is a master regulator of MHC class II gene expression as well as of some of the genes involved in MHC class II antigen presentation. It has recently been discovered that another member of the NLR protein family, NLRC5, transcriptionally activates MHC class I genes, and thus acts as "CITA" (MHC class I transactivator), a counterpart to CIITA. In addition to MHC class I genes, NLRC5 can induce the expression of β2M, TAP1 and LMP2, essential components of MHC class I antigen presentation. These findings indicate that NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and MHC class II pathways, respectively.

The therapeutic effectiveness of ursodeoxycholic acid (UDCA) for various autoimmune liver diseases strongly indicates that UDCA possesses immunomodulatory activities. Experimental evidence also supports this notion, since, for example, UDCA has been shown to suppress secretion of IL-2, IL-4, and IFN-{gamma} from activated T lymphocytes, and Ig production from B lymphocytes. To investigate the mechanical background of UDCA-mediated immunomodulation, we asked whether UDCA interacts with the intracellular signal transduction pathway, especially whether it is involved in immunosuppressive glucocorticoid hormone action. For this purpose, we used a cloned Chinese hamster ovary cell line, CHOpMTGR, in which glucocorticoid receptor cDNA was stably integrated. In immunocytochemical analysis, we found that treatment with UDCA promoted the nuclear translocation of the glucocorticoid receptor in a ligand-independent fashion, which was further confirmed by immunoprecipitation assays. Moreover, the translocated glucocorticoid receptor demonstrated sequence-specific DNA binding activity. Transient transfection experiments revealed that treatment of the cells with UDCA marginally enhanced glucocorticoid-responsive gene expression. We also showed that UDCA suppressed IFN-{gamma}-mediated induction of MHC class II gene expression via the glucocorticoid receptor-mediated pathway. Together, UDCA-dependent promotion of translocation of the glucocorticoid receptor may be associated with, at least in part, its immunomodulatory action through glucocorticoid receptor-mediated gene regulation. 68 refs., 8 figs.

Genes of the major histocompatibility complex (MHC) are valuable makers of adaptive genetic variation in evolutionary ecology research, yet the non-classical MHC genes remain largely unstudied in wild vertebrates. In this study, we have characterized the non-classical MHC class II gene, Egeu-DAB4, in the vulnerable Chinese egret (Ciconiiformes, Ardeidae, Egretta eulophotes). Gene expression analyses showed that Egeu-DAB4 gene had a restricted tissue expression pattern, being expressed in seven examined tissues including the liver, heart, kidney, esophagus, stomach, gallbladder, and intestine, but not in muscle. With respect to polymorphism, only one allele of exon 2 was obtained from Egeu-DAB4 using asymmetric PCR, indicating that Egeu-DAB4 is genetically monomorphic in exon 2. Comparative analyses showed that Egeu-DAB4 had an unusual sequence, with amino acid differences suggesting that its function may differ from those of classical MHC genes. Egeu-DAB4 gene was only found in 30.56-36.56 % of examined Chinese egret individuals. Phylogenetic analysis showed a closer relationship between Egeu-DAB4 and the DAB2 genes in nine other ardeid species. These new findings provide a foundation for further studies to clarify the immunogenetics of non-classical MHC class II gene in the vulnerable Chinese egret and other ciconiiform birds.

Although there have been several previous reports of immunohistochemical staining for MHC antigens in muscle biopsies, there appears to be a lack of consensus about its routine use in the diagnostic evaluation of biopsies from patients with suspected inflammatory myopathy. Positive MHC-I staining is nonspecific but is widely used as a marker for inflammatory myopathy, whilst the role of MHC-II staining is not clearly defined. We investigated the sensitivity and specificity of MHC-I and MHC-II immunostaining for the diagnosis of inflammatory myopathy in a large group of biopsies from a single reference laboratory. Positive staining for MHC-I was found to have a high sensitivity in biopsies from patients with inflammatory myopathy but a very low specificity, as it was also common in other non-inflammatory myopathies and neurogenic disorders. On the other hand, MHC-II positivity had a much higher specificity in all major subgroups of inflammatory myopathy, especially inclusion body myositis. The findings indicate that the combination of MHC-I and MHC-II staining results in a higher degree of specificity for the diagnosis of inflammatory myopathy and that in biopsies with inflammation, positive MHC-II staining strongly supports the diagnosis of an immune-mediated myopathy. We recommend that immunohistochemical staining for both MHC-I and MHC-II should be included routinely in the diagnostic evaluation of muscle biopsies from patients with suspected inflammatory myopathy. However, as the sensitivity and interpretation of MHC staining may depend on the technique used, further studies are needed to compare procedures in different centres and develop standardised protocols.

Class I and class II major histocompatibility complex (MHC) gene products are key recognition units in the induction and regulation of the immune response. Expression of class I and class II may be constitutive or inducible by cytokines such as interferon-gamma (IFN-gamma). A key step in the induction of MHC genes is recognition of IFN-gamma by its membrane receptor. The work described here examines the regulation of the occupied IFN-gamma receptor by the cytoskeleton. To do this the authors have used the fungal metabolites dihydrocytochalasin B (DHCB) and cytochalasin D (CD), substances that bind to actin filaments and thereby disrupt the cytoskeleton. The authors have studied the effect of DHCB and CD on IFN-gamma-induced MHC gene expression in 143 B cells, a human osteosarcoma-derived cell line. Herein the authors demonstrate that alterations in the cytoskeleton induced by DHCB and CD can lead to increases in IFN-gamma-induced MHC gene expression. Dihydrocytochalasin B added up to 3 hours after IFN-gamma results in a threefold to sixfold increase in levels of class II mRNA while producing minimal enhancement of class I gene expression. In contrast, glyceraldehyde-3-phosphate dehydrogenase mRNA expression was unaltered by IFN-gamma or by the cytochalasins. The increased amount of class II mRNA can be accounted for by a concomitant increase in transcription rate of this gene. Studies using 125I-IFN-gamma demonstrate that the occupied IFN-gamma receptor associates with a Triton X-100 insoluble fraction of 143 B cells and that DHCB and CD markedly inhibit this association. The results described here provide evidence that is consistent with the hypothesis that the activity of the occupied IFN-gamma receptor may be modulated by interactions with the cytoskeleton of the cell. This receptor may be one of a group of plasma membrane receptors that are sensitive to the action of cytochalasins after ligand binding. Images Figure 1 Figure 2 PMID:1907805

Our understanding of the evolutionary mechanisms generating variation within the highly polymorphic major histocompatibility complex (MHC) genes remains incomplete. Assessing MHC variation across multiple populations, of recent and ancient divergence, may facilitate understanding of geographical and temporal aspects of variation. Here, we applied 454 sequencing to perform a large-scale, comprehensive analysis of MHC class II in the closely related, hybridizing newts, Lissotriton vulgaris (Lv) and Lissotriton montandoni (Lm). Our study revealed an extensive (299 alleles) geographically structured polymorphism. Populations at the southern margin of the Lv distribution, inhabited by old and distinct lineages (southern Lv), exhibited moderate MHC variation and strong population structure, indicating little gene flow or extensive local adaptation. Lissotriton vulgaris in central Europe and the northern Balkans (northern Lv) and almost all Lm populations had a high MHC variation. A much higher proportion of MHC alleles was shared between Lm and northern Lv than between Lm and southern Lv. Strikingly, the average pairwise F(ST) between northern Lv and Lm was significantly lower than between northern and southern Lv for MHC, but not for microsatellites. Thus, high MHC variation in Lm and northern Lv may result from gene flow between species. We hypothesize that the interspecific exchange of MHC genes may be facilitated by frequency-dependent selection. A marginally significant correlation between the MHC and microsatellite allelic richness indicates that demographic factors may have contributed to the present-day pattern of MHC variation, but unequivocal signatures of adaptive evolution in MHC class II sequences emphasize the role of selection on a longer timescale.

MHC class I (MHC-I) molecules are important components of the immune system. Recently MHC-I have been reported to also play important roles in brain development and synaptic plasticity. In this study, we examine the molecular mechanism(s) underlying activity-dependent MHC-I expression using hippocampal neurons. Here we report that neuronal expression level of MHC-I is dynamically regulated during hippocampal development after birth in vivo. Kainic acid (KA) treatment significantly increases the expression of MHC-I in cultured hippocampal neurons in vitro, suggesting that MHC-I expression is regulated by neuronal activity. In addition, KA stimulation decreased the expression of pre- and post-synaptic proteins. This down-regulation is prevented by addition of an MHC-I antibody to KA treated neurons. Further studies demonstrate that calcium-dependent protein kinase C (PKC) is important in relaying KA simulation activation signals to up-regulated MHC-I expression. This signaling cascade relies on activation of the MAPK pathway, which leads to increased phosphorylation of CREB and NF-κB p65 while also enhancing the expression of IRF-1. Together, these results suggest that expression of MHC-I in hippocampal neurons is driven by Ca2+ regulated activation of the MAPK signaling transduction cascade. PMID:26263390

The major function of major histocompatibility complex (MHC) class II molecules is the presentation of peptide antigens to helper T cells. However, when misfolded proteins are associated with MHC class II molecules in the endoplasmic reticulum, they are transported to the cell surface by MHC class II molecules without processing to peptides. Of note, misfolded proteins complexed with MHC class II molecules are specifically recognized by autoantibodies produced in patients with autoimmune diseases such as rheumatoid arthritis and antiphospholipid syndrome. Furthermore, autoantibody binding to misfolded proteins complexed with MHC class II molecules is associated with the susceptibility to autoimmune diseases conferred by each MHC class II allele. Therefore, misfolded proteins rescued from degradation by MHC class II molecules may be recognized as 'neo-self' antigens by the immune system and be involved in the pathogenicity of autoimmune diseases.

Background Computational prediction of major histocompatibility complex class II (MHC-II) binding peptides can assist researchers in understanding the mechanism of immune systems and developing peptide based vaccines. Although many computational methods have been proposed, the performance of these methods are far from satisfactory. The difficulty of MHC-II peptide binding prediction comes mainly from the large length variation of binding peptides. Methods We develop a novel multiple instance learning based method called MHC2MIL, in order to predict MHC-II binding peptides. We deem each peptide in MHC2MIL as a bag, and some substrings of the peptide as the instances in the bag. Unlike previous multiple instance learning based methods that consider only instances of fixed length 9 (9 amino acids), MHC2MIL is able to deal with instances of both lengths of 9 and 11 (11 amino acids), simultaneously. As such, MHC2MIL incorporates important information in the peptide flanking region. For measuring the distances between different instances, furthermore, MHC2MIL explicitly highlights the amino acids in some important positions. Results Experimental results on a benchmark dataset have shown that, the performance of MHC2MIL is significantly improved by considering the instances of both 9 and 11 amino acids, as well as by emphasizing amino acids at key positions in the instance. The results are consistent with those reported in the literature on MHC-II peptide binding. In addition to five important positions (1, 4, 6, 7 and 9) for HLA(human leukocyte antigen, the name of MHC in Humans) DR peptide binding, we also find that position 2 may play some roles in the binding process. By using 5-fold cross validation on the benchmark dataset, MHC2MIL outperforms two state-of-the-art methods of MHC2SK and NN-align with being statistically significant, on 12 HLA DP and DQ molecules. In addition, it achieves comparable performance with MHC2SK and NN-align on 14 HLA DR molecules. MHC2MIL

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHCII-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHCII) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHCII-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHCII-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis. PMID:26537548

Major histocompatibility complex (MHC) class II molecules are required for the presentation of antigenic peptides that are derived predominantly from internalized proteins. The assembly of MHC class II/peptide complexes occurs within endosomal compartments of antigen-presenting cells (APCs). Therefore, for assembly to occur, MHC class II molecules, foreign proteins, and accessory molecules must be sorted to appropriate intracellular sites. My laboratory is trying to understand how proteins are sorted to various antigen-processing compartments as well as to conventional endosomal organelles. Using chimeric marker proteins and a variety of biochemical and genetic approaches, we are addressing the specificity of protein sorting and the mechanisms by which sorting signals are deciphered. By using a similar chimeric protein approach to target endogenous proteins to distinct compartments, we hope to address the role of processing events in each compartment in the generation of MHC class II ligands.

HLA-DM molecules remove invariant (Ii) chain peptides from newly synthesized MHC class II complexes. Their localization may thus delineate compartments, e.g., MIIC, specialized for loading peptides onto class II molecules. In murine A20 B cells, however, DM is not restricted to specialized endosomal class II-containing vesicles (CIIV). Although DM was found in CIIV, it was also found throughout the endocytic pathway, principally in lysosomes devoid of class II molecules. In human lymphoblasts, HLA-DM was found in structures indistinguishable from late endosomes or lysosomes, although in these cells the lysosomes contained MHC class II molecules. Thus, the distribution of HLA-DM does not necessarily identify specialized class II compartments. Many "MIIC" may represent conventional lysosomes that accumulate MHC class II and HLA-DM in a number of cell types.

In the immune system, the major histocompatibility complex (MHC) class I molecules mediate both the innate and adaptive immune responses in vertebrates. There has been a dogma that the central nervous system (CNS) is immune privileged and healthy neurons do not expressMHC class I molecules. However, recent studies have indicated that the expression and non-immunobiologic roles of MHC class I in mammalian CNS. But data referring to humans are scarce. In this study we report the expression and cellular localization of MHC class I in the human fetal, early postnatal and adult hippocampal formation. The expression of MHC class I was very low in the hippocampus at 20 (gestational weeks) GW and slowly increased at 27-33 GW. The gradually increased expression in the somata of some granular cells in dentate gyrus (DG) was observed at 30-33 GW. Whereas, a rapid increase in MHC class I molecules expression was found in the subiculum and it reached high levels at 31-33 GW and maintained at postnatal 55 days. No expression of MHC class I was found in hippocampal formation in adult. MHC class I heavy chain and β2 microglobulin (β2M) showed similar expression in some cells of the hippocampal formation at 30-33 GW. Moreover, MHC class I molecules were mainly expressed in neurons and most MHC class I-expressing neurons were glutamatergic. The temporal and spatial patterns of MHC class I expression appeared to follow gradients of pyramidal neurons maturation in the subiculum at prenatal stages and suggested that MHC class I molecules are likely to regulate neuron maturation. This article is part of a Special Issue entitled Priority to Publish.

Establishing the genetic determinants of niche adaptation by microbial pathogens to specific hosts is important for the management and control of infectious disease. Streptococcus pyogenes is a globally prominent human-specific bacterial pathogen that secretes superantigens (SAgs) as ‘trademark’ virulence factors. SAgs function to force the activation of T lymphocytes through direct binding to lateral surfaces of T cell receptors and class II major histocompatibility complex (MHC-II) molecules. S. pyogenes invariably encodes multiple SAgs, often within putative mobile genetic elements, and although SAgs are documented virulence factors for diseases such as scarlet fever and the streptococcal toxic shock syndrome (STSS), how these exotoxins contribute to the fitness and evolution of S. pyogenes is unknown. Here we show that acute infection in the nasopharynx is dependent upon both bacterial SAgs and host MHC-II molecules. S. pyogenes was rapidly cleared from the nasal cavity of wild-type C57BL/6 (B6) mice, whereas infection was enhanced up to ∼10,000-fold in B6 mice that express human MHC-II. This phenotype required the SpeA superantigen, and vaccination with an MHC –II binding mutant toxoid of SpeA dramatically inhibited infection. Our findings indicate that streptococcal SAgs are critical for the establishment of nasopharyngeal infection, thus providing an explanation as to why S. pyogenes produces these potent toxins. This work also highlights that SAg redundancy exists to avoid host anti-SAg humoral immune responses and to potentially overcome host MHC-II polymorphisms. PMID:24875883

Human cytomegalovirus (HCMV) productively infects CD34(+) progenitor-derived, mature Langerhans-type dendritic cells (matLC) and reduces surface expression of MHC class II complexes (MHCII) by increasing intracellular retention of these molecules. To determine whether HCMV also inhibits MHCIIexpression by other mechanisms, we assessed mRNA levels of the class II transcriptional regulator, CIITA, and several of its target genes in infected matLC. Levels of CIITA, HLA-DRA (DRA) and DRB transcripts, and new DR protein synthesis were compared in mock-infected and HCMV-infected cells by quantitative PCR and pulse-chase immunoprecipitation analyses, respectively. CIITA mRNA levels were significantly lower in HCMV-infected matLC as compared to mock-infected cells. When assessed in the presence of Actinomycin D, the stability of CIITA transcripts was not diminished by HCMV. Analysis of promoter-specific CIITA isoforms revealed that types I, III and IV all were decreased by HCMV, a result that differs from changes after incubation of these cells with lipopolysaccharide (LPS). Exposure to UV-inactivated virus failed to reduce CIITA mRNA levels, implicating de novo viral gene expression in this effect. HCMV-infected matLC also expressed lower levels of DR transcripts and reduced DR protein synthesis rates compared to mock-infected matLC. In summary, we demonstrate that HCMV infection of a human dendritic cell subset inhibits constitutive CIITA expression, most likely at the transcriptional level, resulting in reduced MHCII biosynthesis. We suggest this represents a new mechanism of modulation of mature LC by HCMV.

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class IIexpressing cells in the sow endometrium following post-ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15-20 h after ovulation [experiment 1, slaughtered at 20-25 h (5-6 h after artificial insemination (AI), group 1-A, n = 4), at 70 h after ovulation (group 1-B, n = 4), on day 11 (group 1-C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1-D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5-6 h after AI (group 2-A, n = 4) or on day 19 (group 2-D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1-A, only one sow had spermatozoa in the utero-tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1-B, altogether 23 of 48 oocytes were cleaved. Day 11 (1-C), embryos with small diameter were observed. Day 19 (1-D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1-A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2-A, no sow had spermatozoa in the UTJ or in the uterine flushings. At

Four different isoforms of the human invariant chain (Ii) have been described (p33, p35, p41 and p43). These heterotrimerize in the endoplasmic reticulum (ER) before associating with MHC class II molecules (MHCIIs). However, the final stoichiometry of the Ii/MHCII complex remains debated. This is particularly interesting as both p35 and p43 include a di-arginine motif that requires masking by MHCII to allow ER egress. Here, to functionally address the requirement for stoichiometric interactions, we used a recombinant DR heterodimer bearing its own cytoplasmic di-lysine ER-retention motif (DRKKAA). When coexpressed with p33 and a control myc-tagged DR (DRmyc), DRKKAA was retained in the ER but had little impact on surface expression of DRmyc. However, when coexpressed with p35, DRKKAA restricted the surface expression of DRmyc, indicating that Ii trimers can be loaded with more than one MHCII. Similar results were obtained using HLA-DQ instead of DRmyc, showing that a single trimeric Ii scaffold can include distinct MHCII isotypes. Altogether, these results demonstrate that the subunit stoichiometry of oligomeric Ii/MHCII complexes is influenced by p35.

In previous studies we have shown down-regulation of class I major histocompatibility complex (MHC) expression in a significant proportion of primary cervical carcinomas, which was found to be strongly correlated with loss of expression of the transporter associated with antigen presentation (TAP). By contrast, class IIMHCexpression was frequently up-regulated on neoplastic keratinocytes in these malignancies. In order to investigate whether these changes are associated with biological behaviour of the tumours, 20 cervical carcinomas were analyzed for MHC (HLA-A, HLA-B/C, HLA-DR) and TAP-1 expression in the primary tumours and in lymph node metastases by immunohistochemistry. The results showed a significant increase in the prevalence of HLA-A and HLA-B/C down-regulation in metastasised neoplastic cells as compared with the primary tumour (P = 0.01). In all cases this was accompanied by loss of TAP-1 expression. Up-regulated HLA-DR expression was found exclusively in primary tumours and was absent in the corresponding metastases (P = 0.002). These data are consistent with the hypothesis that loss of TAP-1 and the consequent down-regulation of class I MHCexpression provides a selective advantage for neoplastic cervical cells during metastasis. Furthermore, the lack of class IIMHCexpression in metastasised cells either reflects a different local lymphokine production or indicates that these cells may have escaped CD4+ cytotoxic T-lymphocyte (CTL)-mediated killing. Images Figure 1 Figure 2 Figure 3 PMID:8198988

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal. PMID:25808889

The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) can cause precipitous population declines in its amphibian hosts. Responses of individuals to infection vary greatly with the capacity of their immune system to respond to the pathogen. We used a combination of comparative and experimental approaches to identify major histocompatibility complex class II (MHC-II) alleles encoding molecules that foster the survival of Bd-infected amphibians. We found that Bd-resistant amphibians across four continents share common amino acids in three binding pockets of the MHC-II antigen-binding groove. Moreover, strong signals of selection acting on these specific sites were evident among all species co-existing with the pathogen. In the laboratory, we experimentally inoculated Australian tree frogs with Bd to test how each binding pocket conformation influences disease resistance. Only the conformation of MHC-II pocket 9 of surviving subjects matched those of Bd-resistant species. This MHC-II conformation thus may determine amphibian resistance to Bd, although other MHC-II binding pockets also may contribute to resistance. Rescuing amphibian biodiversity will depend on our understanding of amphibian immune defence mechanisms against Bd. The identification of adaptive genetic markers for Bd resistance represents an important step forward towards that goal.

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class IIMHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the

Highly polymorphic genes with central roles in lymphocyte mediated immune surveillance are grouped together in the major histocompatibility complex (MHC) in higher vertebrates. Generally, across vertebrate species the class IIMHC DRA gene is highly conserved with only limited allelic variation. Here however, we provide evidence of trans-species polymorphism at the DRA locus in domestic sheep (Ovis aries). We describe variation at the Ovar-DRA locus that is far in excess of anything described in other vertebrate species. The divergent DRA allele (Ovar-DRA*0201) differs from the sheep reference sequences by 20 nucleotides, 12 of which appear non-synonymous. Furthermore, DRA*0201 is paired with an equally divergent DRB1 allele (Ovar-DRB1*0901), which is consistent with an independent evolutionary history for the DR sub-region within this MHC haplotype. No recombination was observed between the divergent DRA and B genes in a range of breeds and typical levels of MHC class II DR protein expression were detected at the surface of leukocyte populations obtained from animals homozygous for the DRA*0201, DRB1*0901 haplotype. Bayesian phylogenetic analysis groups Ovar-DRA*0201 with DRA sequences derived from species within the Oryx and Alcelaphus genera rather than clustering with other ovine and caprine DRA alleles. Tests for Darwinian selection identified 10 positively selected sites on the branch leading to Ovar-DRA*0201, three of which are predicted to be associated with the binding of peptide antigen. As the Ovis, Oryx and Alcelaphus genera have not shared a common ancestor for over 30 million years, the DRA*0201 and DRB1*0901 allelic pair is likely to be of ancient origin and present in the founding population from which all contemporary domestic sheep breeds are derived. The conservation of the integrity of this unusual DR allelic pair suggests some selective advantage which is likely to be associated with the presentation of pathogen antigen to T-cells and the

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II- invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process. PMID:8603911

The chimpanzee (Pan troglodytes, Patr) is the closest zoological living relative of humans and shares approximately 98.6% genetic homology to human beings. Although major histocompatibility complex (MHC) plays a critical role in T cell-mediated immune responses in vertebrates, the information on Patr MHC remains at a relatively poor level. Therefore, we attempted to isolate Patr MHC class II genes and determine their nucleotide sequences. The cDNAs encoding Patr MHC class II DP, DQ and DR beta chains were isolated from the cDNA library of a chimpanzee B lymphocyte cell line Bch261. As a result of screening, the clone 6-3-1 as a representative of Patr DP clone, clone 30-1 as a Patr DQ clone, and clones 4-7-1 and 55-1 having different sequences as Patr DR clones were detected. The clone 6-3-1 consisted of 1,062 nucleotides including an open reading frame (ORF) of 777 bp. In the same way, clone 30-1 consisted of 1,172 nucleotides including ORF of 786 bp, clones 4-7-1 and 55-1 consisted of 1,163 nucleotides including ORF of 801 bp. Except for five nucleotide changes, clones 4-7-1 and 55-1 were the same sequence. By comparison with the nucleotide sequences already reported on chimpanzee MHC class II beta 1 genes, clones 6-3-1, 30-1, 4-7-1 and 55-1 were classified as PatrDPB1*16, PatrDQB1*0302, PatrDRB1*0201 and PatrDRB1*0204, respectively. This is the first report to describe complete cDNA sequences of Patr DP and DQ molecules. The nucleotide sequence data of Patr MHC class II genes obtained in this study will be useful for the genotyping of Patr MHC class II genes in individual chimpanzees.

Direct identification and isolation of antigen-specific T cells became possible with the development of “MHC tetramers”, based on fluorescent avidins displaying biotinylated peptide-MHC (pMHC) complexes. This approach, extensively used for MHC class I–restricted T cells, has met very limited success with MHC class II tetramers (pMHCT-2) for the detection of CD4+ specific T cells. In addition, a very large number of these reagents while capable of specifically activating T cells after being coated on solid support, are still unable to stain. In order to try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-Ad-OVA system as a model. Through this process the geometry of pMHC display by avidin tetramers was examined, as well as the stability of recombinant MHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 repertoire and help us in the production and testing of new vaccines. PMID:19923463

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo

Professional antigen-presenting cells secrete major histocompatibility complex class II (MHCII) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHCII at lysosomally targeted multivesicular bodies, sorting of MHCII into exosomes does not rely on MHCII ubiquitination. In search for proteins that drive the incorporation of MHCII into exosomes or functionally discriminate exosomal from plasma membrane MHCII, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHCII from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3ɛ, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHCII.

TLR-mediated recognition of microbial danger induces substantial changes in macrophage migration, adherence and phagocytosis. Recently, we described the LPS-regulated phosphorylation of many cytoskeleton-associated proteins by phosphoproteomics. The functional role of these cytoskeletal and motor proteins in innate immune cell responses is largely unexplored. Here, we first identified both long-tailed class I myosins Myo1e and Myo1f as important contributors to LPS-triggered macrophage spreading. Mouse bone marrow-derived macrophages and dendritic cells (DCs) deficient in Myo1e selectively secreted increased amounts of the chemokine CCL2. In addition, the cell surface expression of MHC class II (MHC-II) on both cell types was reduced in the absence of Myo1e. However, transcriptional changes in CCL2 and MHC-II were not observed in the absence of Myo1e, indicating that Myo1e regulates specific intracellular transport processes. The capacity of macrophages and DCs lacking Myo1e to stimulate antigen-specific CD4+ T-cell proliferation was impaired, consistent with the reduced MHC-II surface protein levels. Surprisingly, in Myo1e-deficient DCs, the proteolytic cleavage of endocytosed antigen was also increased. Together, our results provide evidence for a non-redundant function of the motor protein Myo1e in the regulation of TLR4-controlled, cytoskeleton-associated functional properties of macrophages and DCs, and in induction of a full MHC-II-restricted adaptive immune response. PMID:25263281

Contrary to neutral markers, the major histocompatibility complex (MHC) can reflect the fitness and adaptive potential of a given species due to its association with the immune system. For this reason, the use of MHC in endangered wildlife management has increased greatly in recent years. Here, we isolated complementary DNA (cDNA) and genomic DNA (gDNA) sequences to characterize the MHC class II β genes in Hainan Eld's deer (Cervus eldi hainanus), a highly endangered cervid, which recovered from a severe population bottleneck consisting of 26 animals. Analysis of 7 individuals revealed the presence of 3 DRB and 3 DQB putatively functional gDNA sequences. The Ceel-DRB and DQB sequences displayed high variability in exon 2, and most nonsynonymous substitutions were detected in this region. Phylogenetic analysis indicated that trans-species evolution of MHC class II β might occur in the Cervinae subfamily. Comparison of the number of sequences between gDNA and cDNA revealed that all sequences isolated from the genome were detectable in the cDNA libraries derived from different tissues (including the liver, kidney, and spleen), suggesting none of these sequences were derived from silent genes or pseudogenes. Characterization of the MHC class II β genes may lay the foundation for future studies on genetic structure, mate choice, and viability analysis in Hainan Eld's deer.

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells. PMID:25606683

MARCH-I (membrane-associated RING-CH I) has been suggested as a physiological E3 ubiquitin ligase for both MHC class II (MHCII) and B7-2. In this study, we show that MARCH-I-mediated MHCII ubiquitination is necessary for the maintenance of conventional dendritic cell (cDC) functions in the steady state. MARCH-I-deficient cDCs accumulated MHCII and B7-2 and exhibited low Ag-presenting ability for exogenous Ags and low cytokine-producing ability upon stimulation in vivo. Importantly, MHCII, but not B7-2, was required for impaired cDC function induced by loss of MARCH-I in vivo. Moreover, MHCII knockin mice whose MHCII was not ubiquitinated showed dysfunction of cDC similar to that of MARCH-I knockout mice. These results suggest that the accumulation of MHCII resulting from loss of ubiquitination caused cDC abnormality; therefore, MARCH-I may function as a housekeeper of cDC in the steady state.

The MHC class I (MHC-I) region in New World monkeys (Platyrrhini) has remained relatively understudied. To evaluate the diversification patterns and transcription behavior of MHC-I in Platyrrhini, we first analyzed public genomic sequences from the MHC-G-like subregion in Saimiri boliviensis, Ateles geoffroyi and Callicebus moloch, and from the MHC-B subregion in Saimiri boliviensis. While S. boliviensis showed multiple copies of both MHC-G-like (10) and –B (15) loci, A. geoffroyi and C. moloch had only three and four MHC-G-like genes, respectively, indicating that not all Platyrrhini species have expanded their MHC-I loci. We then sequenced MHC-G-like and -B cDNAs from nine Platyrrhini species, recovering two to five unique cDNAs per individual for both loci classes. In two Saguinus species, however, no MHC-B cDNAs were found. In phylogenetic trees, MHC-G-like cDNAs formed genus-specific clusters whereas the MHC-B cDNAs grouped by Platyrrhini families, suggesting a more rapid diversification of the former. Furthermore, cDNA sequencing in 12 capuchin monkeys showed that they transcribe at least four MHC-G-like and five MHC-B polymorphic genes, showing haplotypic diversity for gene copy number and signatures of positive natural selection at the peptide binding region. Finally, a quantitative index for MHC:KIR affinity was proposed and tested to predict putative interacting pairs. Altogether, our data indicate that i) MHC-I genes has expanded differentially among Platyrrhini species, ii) Callitrichinae (tamarins and marmosets) MHC-B loci have limited or tissue-specific expression, iii) MHC-G-like genes have diversified more rapidly than MHC-B genes, and iv) the MHC-I diversity is generated mainly by genetic polymorphism and gene copy number variation, likely promoted by natural selection for ligand binding. PMID:26121030

The study describes >400 major histocompatibility complex (MHC) class II B exon 2 and 114 intron 2 sequences of 36 passerine bird species, 13 of which belong to the group of Darwin’s finches (DFs) and the remaining 23 to close or more distant relatives of DFs in Central and South America. The data set is analyzed by a combination of judiciously selected statistical methods. The analysis reveals that reliable information concerning MHC organization, including the assignment of sequences to loci, and evolution, as well as the process of species divergence, can be obtained in the absence of genomic sequence data, if the analysis is taken several steps beyond the standard phylogenetic tree construction approach. The main findings of the present study are these: The MHC class II B region of the passerine birds is as elaborate in its organization, divergence, and genetic diversity as the MHC of the eutherian mammals, specifically the primates. Hence, the reported simplicity of the fowl MHC is an oddity. With the help of appropriate markers, the divergence of the MHC genes can be traced deep in the phylogeny of the bird taxa. Transspecies polymorphism is rampant at many of the bird MHC loci. In this respect, the DFs behave as if they were a single, genetically undifferentiated population. There is thus far no indication of alleles that could be considered species, genus, or even DF group specific. The implication of these findings is that DFs are in the midst of adaptive radiations, in which morphological differentiation into species is running ahead of genetic differentiation in genetic systems such as the MHC or the mitochondrial DNA. The radiations are so young that there has not been enough time to sort out polymorphisms at most of the loci among the morphologically differentiating species. These findings parallel those on Lake Victoria haplochromine fishes. Several of the DF MHC allelic lineages can be traced back to the MHC genes of the species Tiaris obscura

In contrast to rhesus monkeys, substantial knowledge on cynomolgus monkey major histocompatibility complex (MHC) class II haplotypes is lacking. Therefore, 17 animals, including one pedigreed family, were thoroughly characterized for polymorphic Mhc class II region genes as well as their mitochondrial DNA (mtDNA) sequences. Different cynomolgus macaque populations appear to exhibit unique mtDNA profiles reflecting their geographic origin. Within the present panel, 10 Mafa-DPB1, 14 Mafa-DQA1, 12 Mafa-DQB1, and 35 Mafa-DRB exon 2 sequences were identified. All of these alleles cluster into lineages that were previously described for rhesus macaques. Moreover, about half of the Mafa-DPB1, Mafa-DQA1, and Mafa-DQB1 alleles and one third of the Mafa-DRB exon 2 sequences are identical to rhesus macaque orthologues. Such a high level of Mhc class II allele sharing has not been reported for primate species. Pedigree analysis allowed the characterization of nine distinct Mafa class II haplotypes, and seven additional ones could be deduced. Two of these haplotypes harbor a duplication of the Mafa-DQB1 locus. Despite extensive allele sharing, rhesus and cynomolgus monkeys do not appear to possess identical Mhc class II haplotypes, thus illustrating that new haplotypes were generated after speciation by recombination-like processes.

The status of major histocompatibility complex (MHC) class I and II gene expression in the normal mouse lens was examined. No mRNA for either class I or II genes was detectable in mouse lens, while the expression of MHC genes in other tissues generally matched immunohistochemical data from human tissues. However it was observed that MHC class I mRNA is present in the mouse lens-derived cell line alpha TN4-1. From a new-born mouse lens cDNA library a clone was obtained for the murine homologue of the DNA-binding protein dbpB/YB-1, a protein originally identified in human lymphocytes and proposed to be a negative regulator of MHC class II gene expression. Northern blots detect dbpB/YB-1 mRNA in all mouse tissues and cells examined, including both mouse lens and alpha TN4-1 cells, suggesting that dbpB/YB-1 has a general and widespread role.

Initial adhesive contacts between T lymphocytes and dendritic cells (DCs) facilitate recognition of peptide-MHC complexes by the TCR. In this report, we studied the dynamic behavior of adhesion and Ag receptors on DCs during initial contacts with T-cells. Adhesion molecules LFA-1- and ICAM-1,3-GFP as well as MHC class II-GFP molecules were very rapidly concentrated at the DC contact area. Binding of ICAM-3, and ICAM-1 to a lesser extent, to LFA-1 expressed by mature but not immature DC, induced MHC-II clustering into the immune synapse. Also, ICAM-3 binding to DC induced the activation of the Vav1-Rac1 axis, a regulatory pathway involved in actin cytoskeleton reorganization, which was essential for MHC-II clustering on DCs. Our results support a model in which ICAM-mediated MHC-II clustering on DC constitutes a priming mechanism to enhance antigen presentation to T-cells. PMID:15872088

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

The BCR binds antigen for processing and subsequent presentation on MHCII molecules. Polyvalent antigen induces BCR clustering and targeting to endocytic processing compartments, which are also accessed by Ii-MHCII. Here, we report that clustered BCR is able to team up with Ii-MHCII already at the plasma membrane of mouse B-lymphocytes. Colocalization of BCR and Ii-MHCII on the cell surface required clustering of both types of molecules. The clustering of only one type did not trigger the recruitment of the other. Ii-bound MIF (a ligand of Ii) also colocalized with clustered BCR upon oligomerization of MIF on the surface of the B cell. Abundant surface molecules, such as B220 or TfnR, did not cocluster with the BCR. Some membrane raft-associated molecules, such as peptide-loaded MHCII, coclustered with the BCR, whereas others, such as GM1, did not. The formation of a BCR- and Ii-MHCII-containing membrane domain by antibody-mediated clustering was independent of F-actin and led to the coendocytosis of its constituents. With a rapid Brij 98 extraction method, it was possible to capture this membrane domain biochemically as a DRM. Ii and clustered BCR were present on the same DRM, as shown by immunoisolation. The coalescence of BCR and Ii-MHCII increased tyrosine phosphorylation, indicative of enhanced BCR signaling. Our work suggests a novel role for MIF and Ii-MHCII in BCR-mediated antigen processing.

The major histocompatibility complex (MHC) has an important role in immune response and is known as the most polymorphic locus in vertebrates. We developed three pairs of polymerase chain reaction primers of the alpha-2 domain (exon 3) of MHC class I and the beta-2 (exon 3) and beta-3 domains (exon 4) of MHC class II B gene in the German mirror common carp (Cyprinus carpio L.). We analyzed the three loci in a population of 65 individuals that had suffered the serious disease of gill rot. Five to six variable nucleotide sites and two to six variable amino acid sites (71.43%) were detected in the exon sequence of the sampled populations, indicating that many of them corresponded to amino acids involved in antigen recognition. Deviation from Hardy-Weinberg equilibrium and linkage disequilibrium were differentially found in some loci, which will be important for further study of disease resistance/susceptibility and population evolution.

Kin recognition can drive kin selection and the evolution of social behaviour. In zebrafish (Danio rerio, Hamilton 1822), kin recognition is based on olfactory and visual imprinting processes. If larvae are exposed to visual and chemical cues of kin at day 5 and 6 post fertilization they will recognize kin throughout life, while exposure to non-kin fails to trigger any recognition. Chemical imprinting signals are transcribed by polymorphic genes of the major histocompatibility complex (MHC) code; however, the underlying mechanism for visual imprinting remains unclear. Here we provide evidence for the existence of family-specific differences in morphometry and pigmentation pattern of six day old zebrafish larvae. While rump, tail and body pigmentation were dependent on relatedness, iris pigmentation and morphometry were also influenced by MHC class II genotype. Our study revealed that the MHC not only influences the chemical signature of individuals, but also their visual appearance.

Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to accumulate greater amounts of a specific peptide–MHCII (pMHCII), suggesting that MHCII turnover facilitates the replacement of old complexes. We propose that pMHCII complexes are periodically targeted for degradation in centroblasts to favor the presentation of recently acquired antigens, thereby promoting the fidelity and efficiency of selection. PMID:27162138

The critical role of major histocompatibility complex (MHC) genes in disease resistance, along with their putative function in sexual selection, reproduction and chemical ecology, make them an important genetic system in evolutionary ecology. Studying selective pressures acting on MHC genes in the wild nevertheless requires population-wide genotyping, which has long been challenging because of their extensive polymorphism. Here, we report on large-scale genotyping of the MHC class II loci of the grey mouse lemur (Microcebus murinus) from a wild population in western Madagascar. The second exons from MHC-DRB and -DQB of 772 and 672 individuals were sequenced, respectively, using a 454 sequencing platform, generating more than 800,000 reads. Sequence analysis, through a stepwise variant validation procedure, allowed reliable typing of more than 600 individuals. The quality of our genotyping was evaluated through three independent methods, namely genotyping the same individuals by both cloning and 454 sequencing, running duplicates, and comparing parent-offspring dyads; each displaying very high accuracy. A total of 61 (including 20 new) and 60 (including 53 new) alleles were detected at DRB and DQB genes, respectively. Both loci were non-duplicated, in tight linkage disequilibrium and in Hardy-Weinberg equilibrium, despite the fact that sequence analysis revealed clear evidence of historical selection. Our results highlight the potential of 454 sequencing technology in attempts to investigate patterns of selection shaping MHC variation in contemporary populations. The power of this approach will nevertheless be conditional upon strict quality control of the genotyping data.

Genetic polymorphism of the major histocompatibility complex (MHC) B-LB II gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the genetic variation of the B-LB II gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB II sequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification of 31 novel MHC B-LB II alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LB II gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LB II alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were estimated to be 90.6%-99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%+/-0.94% and 14.64%+/-2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the beta1 domain coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specificity of MHC B-LB II molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

In so-called model species, such as human and mouse, genes of the major histocompatibility complex (MHC) are characterized by extremely high levels of polymorphism, and it is considered that such diversity is maintained by balancing selection. ;There is now a recognized need to expand studies into nonmodel species to examine whether high MHC diversity is mirrored in natural populations, and to determine the ecological, ethological, and evolutionary processes that underpin balancing selection. To address such issues, a necessary prerequisite is the ability to characterize diversity at a single, expressed, polymorphic MHC locus on which selection may be acting. Here, we provide the first description of allelic diversity at exon 2 of an MHC class II DRB locus in the European water vole (Arvicola terrestris), characterize variation across four natural populations, and test whether the patterns of variation are consistent with the effects of balancing selection. Using single-strand conformation polymorphism analysis and subsequent DNA sequencing of gel excisions, five DRB alleles were resolved, each with a unique amino acid sequence, among 100 individuals from four geographically distinct populations. Reverse transcription polymerase chain reaction confirmed that the alleles were products from an expressed locus. Intra-allelic amino acid differences were high (10.5-33.3%), and the nonsynonymous substitution rate exceeded the synonymous substitution rate for the functional peptide-binding region (d (N):d (S)=3.91 and P<0.005). Phylogenetic comparison of resolved alleles with closely related homologues indicated that each allele represented a unique lineage preserved across speciation events. These results indicate that balancing selection has maintained diversity of DRB allelic lineages and amino acid function over evolutionary time scales, but may be less effective at preserving alleles in contemporary populations where stochastic microevolutionary processes may

Tumor or metastatic cells lose MHC class I (MHC-I) expression during cancer progression as an escape mechanism from immune surveillance. These defects in MHC-I may be reversible by cytokines or different agents (soft lesions) or irreversible due to structural defects (hard lesions). The nature of these MHC-I alterations might determine the success or failure of immunotherapy treatments. In this study, we have used an MHC-I-positive murine fibrosarcoma tumor clone, GR9-A7, which generates multiple lung and lymph node metastases with reversible MHC-I alterations after treatment with IFN-γ. Four different antitumor treatments were carried out after primary tumor excision to determine their capacity to inhibit spontaneous metastatic colonization of the GR9-A7 tumor clone. We found that 2 different immunotherapy protocols (CpG plus autologous irradiated-GR9-A7 cells and protein-bound polysaccharide K (PSK) and 1 chemoimmunotherapy (docetaxel plus PSK) induced eradication of metastases. In contrast, chemotherapy with docetaxel alone produced only partial reduction in the number of metastases. Flow cytometric analysis of lymphocyte populations showed an immunosuppression in GR9-A7 tumor-bearing host, which could be reverted by immunotherapy treatments. Our results suggest that irreversible or reversible MHC-I alterations in tumor target cells may determine its progression or regression independently of the type of immunotherapy used.

Dysregulated protein phosphorylation is a hallmark of malignant transformation. Transformation can generate major histocompatibility complex (MHC)-bound phosphopeptides that are differentially displayed on tumor cells for specific recognition by T cells. To understand how phosphorylation alters the antigenic identity of self-peptides and how MHC class II molecules present phosphopeptides for CD4{sup +} T-cell recognition, we determined the crystal structure of a phosphopeptide derived from melanoma antigen recognized by T cells-1 (pMART-1), selectively expressed by human melanomas, in complex with HLA-DR1. The structure revealed that the phosphate moiety attached to the serine residue at position P5 of pMART-1 is available for direct interactions with T-cell receptor (TCR) and that the peptide N-terminus adopts an unusual conformation orienting it toward TCR. This structure, combined with measurements of peptide affinity for HLA-DR1 and of peptide-MHC recognition by pMART-1-specific T cells, suggests that TCR recognition is focused on the N-terminal portion of pMART-1. This recognition mode appears to be distinct from that of foreign antigen complexes but is remarkably reminiscent of the way autoreactive TCRs engage self- or altered self-peptides, consistent with the tolerogenic nature of tumor-host immune interactions.

The mammalian fetus represents a semiallograft within the maternal uterus yet is not rejected. This situation is particularly pronounced in species with a hemochorial type of placentation, such as humans and rodents, where maternal tissues and blood are in direct contact with fetal trophoblast and thus potentially with paternal antigens. The main polymorphic antigens responsible for graft rejection are MHC antigens. In humans the trophoblast cells invading into the decidua have a unique pattern of MHC class I expression characterized by both classical (HLA-C) and nonclassical (HLA-G and HLA-E) molecules. Whether such an unusual MHC repertoire on the surface of trophoblast is a conserved feature between species with hemochorial placentation has not been resolved. Here we demonstrate, using a range of methods, that C57BL/6 mouse trophoblast predominantly expresses only one MHC class I antigen, H2-K, at the cell surface of giant cells but lacks expression of nonclassical MHC molecules. Antigenic disparity between parental MHCs affects trophoblast-induced transformation of the uterine vasculature and, consequently, placental and fetal gowth. Maternal uterine blood vessels were more dilated, allowing for increased blood supply, in certain combinations of maternal and paternal MHC haplotypes, and these allogeneic fetuses and placentas were heavier at term compared with syngeneic controls. Thus, maternal-fetal immune interactions are instrumental to optimize reproductive success. This cross-talk has important implications for human disorders of pregnancy, such as preeclampsia and fetal growth restriction.

Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and IIMHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

Genes of the major histocompatibility complex (MHC) encode receptor molecules that are responsible for recognition of intracellular and extracellular pathogens (class I and class II genes, respectively) in vertebrates. Given the different roles of class I and IIMHC genes, one might expect the strength of selection to differ between these two classes. Different selective pressures may also promote different rates of gene conversion at each class. Despite these predictions, surprisingly few studies have looked at differences between class I and II genes in terms of both selection and gene conversion. Here, we investigated the molecular evolution of MHC class I and II genes in five closely related species of prairie grouse (Centrocercus and Tympanuchus) that possess one class I and two class II loci. We found striking differences in the strength of balancing selection acting on MHC class I versus class II genes. More than half of the putative antigen-binding sites (ABS) of class II were under positive or episodic diversifying selection, compared with only 10% at class I. We also found that gene conversion had a stronger role in shaping the evolution of MHC class II than class I. Overall, the combination of strong positive (balancing) selection and frequent gene conversion has maintained higher diversity of MHC class II than class I in prairie grouse. This is one of the first studies clearly demonstrating that macroevolutionary mechanisms can act differently on genes involved in the immune response against intracellular and extracellular pathogens.

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.

Many human solid tumors expressMHCII molecules, and proteins normally localized to melanosomes give rise to MHCII restricted epitopes in melanoma. However, the pathways by which this occurs have not been defined. We analyzed the processing of one such epitope, gp10044-59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1hi/MHCII+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044-59 presentation. By depletion of the AP2 adaptor protein using siRNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044-59 epitope production. Gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHCII molecules. Gp10044-59 presentation is dramatically reduced, and processing occurs entirely in early endosomes / stage I melanosomes. This suggests that melanosomes are inefficient antigen processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHCII restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition. PMID:19017974

Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

Most skin pathologies are characterized by unbalanced synthesis of major histocompatability complex II (MHC-II) proteins. Healthy skin keratinocytes simultaneously produce large amounts of MHC-II and regeneration-supporting proteins, e.g. erythropoietin (EPO), EPO receptor (EPOR), glutamine synthetase (GS) and metallothionein (MT). To investigate the level of regeneration-supporting proteins in the skin during misbalanced production of MHC-II, skin sections from nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gamma (c) (null) and or Foxn1 nu/nu mice which are a priory known to under- and over-expressMHCII, respectively, were used. Double immunofluorescence analysis of NOD/SCID/gamma (c) (null) skin sections showed striking decrease in expression of MHC-II, EPO, GS and MT. In Foxn1 nu/nu mouse skin, GS was strongly expressed in epidermis and in hair follicles (HF), which lacked EPO. In nude mouse skin EPO and MHC-II were over-expressed in dermal fibroblasts and they were completely absent from cortex, channel, medulla and keratinocytes surrounding the HF, suggest a role for EPO in health and pathology of hair follicle. The level of expression of EPO and GS in both mutant mice was confirmed by results of Western blot analyses. Strong immunoresponsiveness of EPOR in the hair channels of NOD/SCID/gamma (c) (null) mouse skin suggests increased requirements of skin cells for EPO and possible benefits of exogenous EPO application during disorders of immune system accompanied by loss MHC-II in skin cells.

The common marmoset (Callithrix jacchus) is a New World primate species that is highly susceptible to fatal infections caused by various strains of bacteria. We present here a first step in the molecular characterization of the common marmoset's Mhc class II genes by nucleotide sequence analysis of the polymorphic exon 2 segments. For this study, genetic material was obtained from animals bred in captivity as well as in the wild. The results demonstrate that the common marmoset has, like other primates, apparently functional Mhc-DR and -DQ regions, but the Mhc-DP region has been inactivated. At the -DR and -DQ loci, only a limited number of lineages were detected. On the basis of the number of alleles found, the -DQA and -B loci appear to be oligomorphic, whereas only a moderate degree of polymorphism was observed for two of three Mhc-DRB loci. The contact residues in the peptide-binding site of the Caja-DRB1*03 lineage members are highly conserved, whereas the -DRB*W16 lineage members show more divergence in that respect. The latter locus encodes five oligomorphic lineages whose members are not observed in any other primate species studied, suggesting rapid evolution, as illustrated by frequent exchange of polymorphic motifs. All common marmosets tested were found to share one monomorphic type of Caja-DRB*W12 allele probably encoded by a separate locus. Common marmosets apparently lack haplotype polymorphism because the number of Caja-DRB loci present per haplotype appears to be constant. Despite this, however, an unexpectedly high number of allelic combinations are observed at the haplotypic level, suggesting that Caja-DRB alleles are exchanged frequently between chromosomes by recombination, promoting an optimal distribution of limited Mhc polymorphisms among individuals of a given population. This peculiar genetic make up, in combination with the limited variability of the major histocompatability complex class II repertoire, may contribute to the common

The bare lymphocyte syndrome (BLS) is a hereditary immunodeficiency resulting from the absence of major histocompatibility complex class II (MHCII) expression. Considering the central role of MHCII molecules in the development and activation of CD4(+) T cells, it is not surprising that the immune system of the patients is severely impaired. BLS is the prototype of a "disease of gene regulation." The affected genes encode RFXANK, RFX5, RFXAP, and CIITA, four regulatory factors that are highly specific and essential for MHCII genes. The first three are subunits of RFX, a trimeric complex that binds to all MHCII promoters. CIITA is a non-DNA-binding coactivator that functions as the master control factor for MHCII expression. The study of RFX and CIITA has made major contributions to our comprehension of the molecular mechanisms controlling MHCII genes and has made this system into a textbook model for the regulation of gene expression.

In this study, we partly characterize major histocompatibility complex (MHC) class II B in the blue tit (Cyanistes caeruleus). A total of 22 individuals from three different European locations: Spain, The Netherlands, and Sweden were screened for MHC allelic diversity. The MHC genes were investigated using both PCR-based methods and unamplified genomic DNA with restriction fragment length polymorphism (RFLP) and southern blots. A total of 13 different exon 2 sequences were obtained independently from DNA and/or RNA, thus confirming gene transcription and likely functionality of the genes. Nine out of 13 alleles were found in more than one country, and two alleles appeared in all countries. Positive selection was detected in the region coding for the peptide binding region (PBR). A maximum of three alleles per individual was detected by sequencing and the RFLP pattern consisted of 4–7 fragments, indicating a minimum number of 2–4 loci per individual. A phylogenetic analysis, demonstrated that the blue tit sequences are divergent compared to sequences from other passerines resembling a different MHC lineage than those possessed by most passerines studied to date. PMID:23919136

Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2–MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs. PMID:21752831

Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing. PMID:23951115

Mycobacterium tuberculosis is an exceptionally successful human pathogen. A major component of this success is the ability of the bacteria to infect immunocompetent individuals and to evade eradication by an adaptive immune response that includes production of the macrophage-activating cytokine, IFN-gamma. Although IFN-gamma is essential for arrest of progressive tuberculosis, it is insufficient for efficacious macrophage killing of the bacteria, which may be due to the ability of M. tuberculosis to inhibit selected macrophage responses to IFN-gamma. In vitro studies have determined that mycobacterial lipoproteins and other components of the M. tuberculosis cell envelope, acting as agonists for TLR2, inhibit IFN-gamma induction of MHC class II. In addition, M. tuberculosis peptidoglycan and IL-6 secreted by infected macrophages inhibit IFN-gamma induction of MHC class II in a TLR2-independent manner. To determine whether TLR2-dependent inhibition of macrophage responses to IFN-gamma is quantitatively dominant over the TLR2-independent mechanisms in vivo, we prepared mixed bone marrow chimeric mice in which the hemopoietic compartment was reconstituted with a mixture of TLR(+/+) and TLR2(-/-) cells. When the chimeric mice were infected with M. tuberculosis, the expression of MHC class II on TLR2(+/+) and TLR2(-/-) macrophages from the lungs of individual infected chimeric mice was indistinguishable. These results indicate that TLR2-dependent and -independent mechanisms of inhibition of responses to IFN-gamma are equivalent in vivo, and that M. tuberculosis uses multiple pathways to abrogate the action of an important effector of adaptive immunity. This work was supported by National Institutes of Health Grants AI 065357-AI 020010.

DNA techniques such as sequence-specific oligonucleotide probe (SSOP) hybridizations, restriction-fragment length polymorphism (RFLP) analyses, and DNA sequencing have greatly supported the characterization of Mhc class II allelic polymorphism. Here the authors describe a DPA 1 allele which has been identified in two male individuals from Liberia and Benin, West Africa, during a survey study on Mhc class II associations with the different manifestations after infection with Onchocerca volvulus. 4 refs., 1 fig.

The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.

The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle. Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly, such mRNA differences were not evident for any of the genes encoding mitochondrial oxidative proteins, 3-hydroxyacyl dehydrogenase, carnitine palmitoyl transferase I, citrate synthase, alpha-ketogluterate dehydrogenase, and cytochrome c, nor for the transcriptional regulators peroxisome proliferator activator receptor gamma coactivator-1alpha, forkhead box O1, or peroxisome proliferator activator receptor-alpha. Thus the mRNA expression of genes encoding mitochondrial proteins and transcriptional regulators does not seem to be fiber type specific as the genes encoding glycolytic and lipid metabolism genes, which suggests that basal mRNA regulation of genes encoding mitochondrial proteins does not match the wide differences in mitochondrial content of these muscles.

TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2−/− mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-IIexpression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHCIIexpression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4+ T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection. PMID:27917375

When dendritic cells (DCs) encounter signals associated with infection or inflammation, they become activated and undergo maturation. Mature DCs are very efficient at presenting antigens captured in association with their activating signal but fail to present subsequently encountered antigens, at least in vitro. Such impairment of MHC class II (MHCII) antigen presentation has generally been thought to be a consequence of down-regulation of endocytosis, so it might be expected that antigens synthesized by the DCs themselves (for instance, viral antigens) would still be presented by mature DCs. Here, we show that DCs matured in vivo could still capture and process soluble antigens, but were unable to present peptides derived from these antigens. Furthermore, presentation of viral antigens synthesized by the DCs themselves was also severely impaired. Indeed, i.v. injection of pathogen mimics, which caused systemic DC activation in vivo, impaired the induction of CD4 T cell responses against subsequently encountered protein antigens. This immunosuppressed state could be reversed by adoptive transfer of DCs loaded exogenously with antigens, demonstrating that impairment of CD4 T cell responses was due to lack of antigen presentation rather than to overt suppression of T cell activation. The biochemical mechanism underlying this phenomenon was the down-regulation of MHC II–peptide complex formation that accompanied DC maturation. These observations have important implications for the design of prophylactic and therapeutic DC vaccines and contribute to the understanding of the mechanisms causing immunosuppression during systemic blood infections. PMID:17978177

The genome of the grey wolf, one of the most widely distributed land mammal species, has been subjected to both stochastic factors, including biogeographical subdivision and population fragmentation, and strong selection during the domestication of the dog. To explore the effects of drift and selection on the partitioning of MHC variation in the diversification of species, we present nine DQA, 10 DQB, and 17 DRB1 sequences of the second exon for European wolves and compare them with sequences of North American wolves and dogs. The relatively large number of class II alleles present in both European and North American wolves attests to their large historical population sizes, yet there are few alleles shared between these regions at DQB and DRB1. Similarly, the dog has an extensive array of class IIMHC alleles, a consequence of a genetically diverse origin, but allelic overlap with wolves only at DQA. Although we might expect a progression from shared alleles to shared allelic lineages during differentiation, the partitioning of diversity between wolves and dogs at DQB and DRB1 differs from that at DQA. Furthermore, an extensive region of nucleotide sequence shared between DRB1 and DQB alleles and a shared motif suggests intergenic recombination may have contributed to MHC diversity in the Canidae.

The expression of MHC class I, MHC class II and the chemokines IP-10, MIP-1alpha, RANTES, fractalkine and I-TAC has been analyzed after intra-vaginal treatment with three synthetic toll-like receptors (TLR) agonists-double-stranded RNA (poly I:C), imiquimod and CpG-rich oligonucleotides (CpG-ODN). These compounds act mainly through TLR3, TLR7 and TLR9, respectively. CpG-ODN induced an accumulation of leucocytes in the vagina, and a strong up-regulation of MHC class I expression on both leucocytes and epithelial cells. Imiquimod and poly I:C induced a weak MHC class I up-regulation in the epithelium but not in the lamina propria. Neither treatment had any profound effect on expression of MHC class II on epithelial cells but poly I:C and to a lesser extent CpG-ODN, up-regulated MHC class II staining intensity which, in the case of CpG-ODN, treatment, was associated with a strong accumulation of CD11c-positive dendritic cells. All three treatments induced an early (8h) but transient IP-10 response. Imiquimod and CpG-ODN, but not poly I:C induced an early MIP-1alpha response which remained for at least 7 days in CpG-ODN-treated animals but not in imiquimod-treated mice. Poly I:C and CpG-ODN, but not imiquimod, induced significant levels of RANTES at different time-points post-treatment. None of the treatments induced any significant changes in the levels of fractalkine, I-TAC or IFN-alpha. These studies have implications for the manipulation of the genital immune response and also improving the outcome of vaginal immunotherapy.

Human leukocyte antigen DM (HLA-DM) molecules are structurally related to classical MHC class II molecules and reside in the lysosome-like compartment where class II-restricted antigen processing is thought to occur. Mutant cell lines lacking HLA-DM are defective in antigen processing and accumulate class II molecules associated with a nested set of invariant chain-derived peptides (class II-associated invariant chain peptides, CLIP). Here we show that HLA-DM catalyzes the dissociation of CLIP from MHC class II-CLIP complexes in vitro and facilitates the binding of antigenic peptides. The reaction has an acidic pH optimum, consistent with its occurrence in a lysosome-like compartment in vivo. Antibody blocking experiments suggest that a transient interaction between HLA-DM and the MHC class II-CLIP complex is required.

WNV is a zoonotic neurotropic flavivirus that has recently emerged globally as a significant cause of viral encephalitis. The last five years, 624 incidents of WNV infection have been reported in Greece. The risk for severe WNV disease increases among immunosuppressed individuals implying thus the contribution of the MHC locus to the control of WNV infection. In order to investigate a possible association of MHC class II genes, especially HLA-DPA1, HLA-DQA1, HLA-DRB1, we examined 105 WNV patients, including 68 cases with neuroinvasive disease and 37 cases with mild clinical phenotype, collected during the period from 2010 to2013, and 100 control individuals selected form the Greek population. Typing was performed for exon 2 for all three genes. DQA1*01:01 was considered to be "protective" against WNV infection (25.4% vs 40.1%, P = 0.004) while DQA1*01:02 was associated with increased susceptibility (48.0% vs 32.1%, P = 0.003). Protection against neuroinvasion was associated with the presence of DRB1*11:02 (4.99% vs 0.0%, P = 0.018). DRB1*16:02 was also absent from the control cohort (P = 0.016). Three additional population control groups were used in order to validate our results. No statistically significant association with the disease was found for HLA-DPA alleles. The results of the present study provide some evidence that MHC class II is involved in the response to WNV infection, outlining infection "susceptibility" and "CNS-high-risk" candidates. Furthermore, three new alleles were identified while the frequency of all alleles in the study was compared with worldwide data. The characterization of the MHC locus could help to estimate the risk for severe WNV cases in a country.

WNV is a zoonotic neurotropic flavivirus that has recently emerged globally as a significant cause of viral encephalitis. The last five years, 624 incidents of WNV infection have been reported in Greece. The risk for severe WNV disease increases among immunosuppressed individuals implying thus the contribution of the MHC locus to the control of WNV infection. In order to investigate a possible association of MHC class II genes, especially HLA-DPA1, HLA-DQA1, HLA-DRB1, we examined 105 WNV patients, including 68 cases with neuroinvasive disease and 37 cases with mild clinical phenotype, collected during the period from 2010 to2013, and 100 control individuals selected form the Greek population. Typing was performed for exon 2 for all three genes. DQA1*01:01 was considered to be "protective" against WNV infection (25.4% vs 40.1%, P = 0.004) while DQA1*01:02 was associated with increased susceptibility (48.0% vs 32.1%, P = 0.003). Protection against neuroinvasion was associated with the presence of DRB1*11:02 (4.99% vs 0.0%, P = 0.018). DRB1*16:02 was also absent from the control cohort (P = 0.016). Three additional population control groups were used in order to validate our results. No statistically significant association with the disease was found for HLA-DPA alleles. The results of the present study provide some evidence that MHC class II is involved in the response to WNV infection, outlining infection "susceptibility" and "CNS-high-risk" candidates. Furthermore, three new alleles were identified while the frequency of all alleles in the study was compared with worldwide data. The characterization of the MHC locus could help to estimate the risk for severe WNV cases in a country. PMID:27812212

Most tumor cells derive from MHC-I-positive normal counterparts and remain positive at early stages of tumor development. T lymphocytes can infiltrate tumor tissue, recognize and destroy MHC class I (MHC-I)-positive cancer cells ("permissive" phase I). Later, MHC-I-negative tumor cell variants resistant to T-cell killing emerge. During this process, tumors first acquire a heterogeneous MHC-I expression pattern and finally become uniformly MHC-I-negative. This stage (phase II) represents a "non-permissive" encapsulated structure with tumor nodes surrounded by fibrous tissue containing different elements including leukocytes, macrophages, fibroblasts, etc. Molecular mechanisms responsible for total or partial MHC-I downregulation play a crucial role in determining and predicting the antigen-presenting capacity of cancer cells. MHC-I downregulation caused by reversible ("soft") lesions can be upregulated by TH1-type cytokines released into the tumor microenvironment in response to different types of immunotherapy. In contrast, when the molecular mechanism of the tumor MHC-I loss is irreversible ("hard") due to a genetic defect in the gene/s coding for MHC-I heavy chains (chromosome 6) or beta-2-microglobulin (B2M) (chromosome 15), malignant cells are unable to upregulate MHC-I, remain undetectable by cytotoxic T-cells, and continue to grow and metastasize. Based on the tumor MHC-I molecular analysis, it might be possible to define MHC-I phenotypes present in cancer patients in order to distinguish between non-responders, partial/short-term responders, and likely durable responders. This highlights the need for designing strategies to enhance tumor MHC-I expression that would allow CTL-mediated tumor rejection.

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (M(PHI)) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-(alpha). These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-(alpha) or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.

Background Research on the genetic architecture and diversity of the MHC has focused mainly on eutherian mammals, birds and fish. So far, studies on model marsupials used in laboratory investigations indicated very little or even no variation in MHC class II genes. However, natural levels of diversity and selection are unknown in marsupials as studies on wild populations are virtually absent. We used two endemic South American mouse opossums, Gracilinanus microtarsus and Marmosops incanus, to investigate characteristic features of MHC selection. This study is the first investigation of MHC selection in free-ranging Neotropical marsupials. In addition, the evolutionary history of MHC lineages within the group of marsupials was examined. Results G. microtarsus showed extensive levels of MHC diversity within and among individuals as 47 MHC-DAB alleles and high levels of sequence divergence were detected at a minimum of four loci. Positively selected codon sites were identified, of which most were congruent with human antigen binding sites. The diversity in M. incanus was rather low with only eight observed alleles at presumably two loci. However, these alleles also revealed high sequence divergence. Again, positive selection was identified on specific codon sites, all congruent with human ABS and with positively selected sites observed in G. microtarsus. In a phylogenetic comparison alleles of M. incanus interspersed widely within alleles of G. microtarsus with four alleles being present in both species. Conclusion Our investigations revealed extensive MHC class II polymorphism in a natural marsupial population, contrary to previous assumptions. Furthermore, our study confirms for the first time in marsupials the presence of three characteristic features common at MHC loci of eutherian mammals, birds and fish: large allelic sequence divergence, positive selection on specific sites and trans-specific polymorphism. PMID:18534008

Hemopoietic stem cell-derived mature Langerhans-type dendritic cells (LC) are susceptible to productive infection by human CMV (HCMV). To investigate the impact of infection on this cell type, we examined HLA-DR biosynthesis and trafficking in mature LC cultures exposed to HCMV. We found decreased surface HLA-DR levels in viral Ag-positive as well as in Ag-negative mature LC. Inhibition of HLA-DR was independent of expression of unique short US2-US11 region gene products by HCMV. Indeed, exposure to UV-inactivated virus, but not to conditioned medium from infected cells, was sufficient to reduce HLA-DR on mature LC, implicating particle binding/penetration in this effect. Reduced surface levels reflected an altered distribution of HLA-DR because total cellular HLA-DR was not diminished. Accumulation of HLA-DR was not explained by altered cathepsin S activity. Mature, peptide-loaded HLA-DR molecules were retained within cells, as assessed by the proportion of SDS-stable HLA-DR dimers. A block in egress was implicated, as endocytosis of surface HLA-DR was not increased. Immunofluorescence microscopy corroborated the intracellular retention of HLA-DR and revealed markedly fewer HLA-DR-positive dendritic projections in infected mature LC. Unexpectedly, light microscopic analyses showed a dramatic loss of the dendrites themselves and immunofluorescence revealed that cytoskeletal elements crucial for the formation and maintenance of dendrites are disrupted in viral Ag-positive cells. Consistent with these dendrite effects, HCMV-infected mature LC exhibit markedly reduced chemotaxis in response to lymphoid chemokines. Thus, HCMV impedes MHC class II molecule trafficking, dendritic projections, and migration of mature LC. These changes likely contribute to the reduced activation of CD4+ T cells by HCMV-infected mature LC.

Background Antigen presenting cells (APCs) sample the extra cellular space and present peptides from here to T helper cells, which can be activated if the peptides are of foreign origin. The peptides are presented on the surface of the cells in complex with major histocompatibility class II (MHCII) molecules. Identification of peptides that bind MHCII molecules is thus a key step in rational vaccine design and developing methods for accurate prediction of the peptide:MHC interactions play a central role in epitope discovery. The MHC class II binding groove is open at both ends making the correct alignment of a peptide in the binding groove a crucial part of identifying the core of an MHC class II binding motif. Here, we present a novel stabilization matrix alignment method, SMM-align, that allows for direct prediction of peptide:MHC binding affinities. The predictive performance of the method is validated on a large MHC class II benchmark data set covering 14 HLA-DR (human MHC) and three mouse H2-IA alleles. Results The predictive performance of the SMM-align method was demonstrated to be superior to that of the Gibbs sampler, TEPITOPE, SVRMHC, and MHCpred methods. Cross validation between peptide data set obtained from different sources demonstrated that direct incorporation of peptide length potentially results in over-fitting of the binding prediction method. Focusing on amino terminal peptide flanking residues (PFR), we demonstrate a consistent gain in predictive performance by favoring binding registers with a minimum PFR length of two amino acids. Visualizing the binding motif as obtained by the SMM-align and TEPITOPE methods highlights a series of fundamental discrepancies between the two predicted motifs. For the DRB1*1302 allele for instance, the TEPITOPE method favors basic amino acids at most anchor positions, whereas the SMM-align method identifies a preference for hydrophobic or neutral amino acids at the anchors. Conclusion The SMM-align method was

MHC class II-restricted antigen presentation requires trafficking of newly synthesized class II-invariant chain complexes from the trans-Golgi network to endosomal, peptide-loading compartments. This transport is mediated by dileucine-like motifs within the cytosolic tail of the invariant chain. Although these signals have been well characterized, the cytosolic proteins that interact with these dileucine signals and mediate Golgi sorting and endosomal transport have not been identified. Recently, an adaptor complex, AP-3, has been identified that interacts with dileucine motifs and mediates endosomal/lysosomal transport in yeast, Drosophila, and mammals. In this report, we have assessed class II-invariant chain trafficking in a strain of mice (mocha) which lacks expression of AP-3. Our studies demonstrate that the lack of AP-3 does not affect the kinetics of invariant chain degradation, the route of class II-invariant chain transport, or the rate and extent of class II-peptide binding as assessed by the generation of SDS-stable dimers. The possible role of other known or unknown adaptor complexes in class II-invariant chain transport is discussed.

The common marmoset (Callithrix jacchus) is a New World primate species that is highly susceptible to fatal infections caused by various strains of bacteria. We present here a first step in the molecular characterization of the common marmoset’s Mhc class II genes by nucleotide sequence analysis of the polymorphic exon 2 segments. For this study, genetic material was obtained from animals bred in captivity as well as in the wild. The results demonstrate that the common marmoset has, like other primates, apparently functional Mhc-DR and -DQ regions, but the Mhc-DP region has been inactivated. At the -DR and -DQ loci, only a limited number of lineages were detected. On the basis of the number of alleles found, the -DQA and -B loci appear to be oligomorphic, whereas only a moderate degree of polymorphism was observed for two of three Mhc-DRB loci. The contact residues in the peptide-binding site of the Caja-DRB1*03 lineage members are highly conserved, whereas the -DRB*W16 lineage members show more divergence in that respect. The latter locus encodes five oligomorphic lineages whose members are not observed in any other primate species studied, suggesting rapid evolution, as illustrated by frequent exchange of polymorphic motifs. All common marmosets tested were found to share one monomorphic type of Caja-DRB*W12 allele probably encoded by a separate locus. Common marmosets apparently lack haplotype polymorphism because the number of Caja-DRB loci present per haplotype appears to be constant. Despite this, however, an unexpectedly high number of allelic combinations are observed at the haplotypic level, suggesting that Caja-DRB alleles are exchanged frequently between chromosomes by recombination, promoting an optimal distribution of limited Mhc polymorphisms among individuals of a given population. This peculiar genetic make up, in combination with the limited variability of the major histocompatability complex class II repertoire, may contribute to the common

The expression of MHC class I molecules is crucial for the initiation and regulation of adaptive immune responses against pathogens. NOD-, LRR- and CARD-containing 5 (NLRC5) was recently identified as a specific transactivator of MHC class I genes (CITA). NLRC5 and the master regulator for MHC class II genes, class II transactivator (CIITA), interact with similar MHC promoter-bound factors. Here, we provide a broad overview of the molecular mechanisms behind MHC class I transcription and the role of the class I transactivator NLRC5 in MHC class I-dependent immune responses.

Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-A{sup g7}, typically have a small, uncharged amino acid residue at position 57 of their {beta} chain ({beta}57); this results in the absence of a salt bridge between {beta}57 and Arg{alpha}76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Arg{alpha}76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3{beta}. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 {angstrom}) electrostatic interactions existed among Arg{alpha}76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue {beta}57 of the I-A{sup g7} and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-A{sub g7}, typically have a small, uncharged amino acid residue at position 57 of their {beta} chain ({beta}57); this results in the absence of a salt bridge between {beta}57 and Arg{alpha}76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Arg{alpha}76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3{beta}. The crystal structure of one such TCR in complex with I-A{sub g7} bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 {angstrom}) electrostatic interactions existed among Arg{alpha}76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue {beta}57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

HLA-DO (H2-O in mice) is an intracellular non-classical MHC class II molecule (MHCII). It forms a stable complex with HLA-DM (H2-M in mice) and shapes the MHC class II-associated peptide repertoire. Here, we tested the impact of HLA-DO and H2-O on the binding of superantigens (SAgs), which has been shown previously to be sensitive to the structural nature of the class II-bound peptides. We found that the binding of staphylococcal enterotoxin (SE) A and B, as well as toxic shock syndrome toxin 1 (TSST-1), was similar on the HLA-DO(+) human B cell lines 721.45 and its HLA-DO(-) counterpart. However, overexpressing HLA-DO in MHC class II(+) HeLa cells (HeLa-CIITA-DO) improved binding of SEA and TSST-1. Accordingly, knocking down HLA-DO expression using specific siRNAs decreased SEA and TSST-1 binding. We tested directly the impact of the class II-associated invariant chain peptide (CLIP), which dissociation from MHC class II molecules is inhibited by overexpressed HLA-DO. Loading of synthetic CLIP on HLA-DR(+) cells increased SEA and TSST-1 binding. Accordingly, knocking down HLA-DM had a similar effect. In mice, H2-O deficiency had no impact on SAgs binding to isolated splenocytes. Altogether, our results demonstrate that the sensitivity of SAgs to the MHCII-associated peptide has physiological basis and that the effect of HLA-DO on SEA and TSST-1 is mediated through the inhibition of CLIP release.

Recognition in the 1980 s that keratinocytes can express class II molecules of the Major Histocompatibility Complex (MHC) first raised the possibility that these cells might have an immunological function, and may even act as antigen presenting cells (APC). For effective T lymphocyte activation, APC require, in addition to MHCII, appropriate costimulatory signals. The aim of this study was to determine the expression of MHC class II and the co-stimulatory molecules CD40, CD80 and CD86 in keratinocytes derived from healthy oral mucosa and oral carcinomas. Using flow cytometry, it was confirmed that oral keratinocytes, switch on, expression of MHC class II molecules after stimulation with IFNgamma in vitro. All keratinocyte lines expressed CD40 constitutively; by contrast, CD80 and CD86 were universally absent. Loss of CD80 and CD86 may be one means whereby tumours escape immunological surveillance.

The largest remaining carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is currently under threat of extinction due to a fatal contagious cancer-devil facial tumour disease. Low major histocompatibility complex (MHC) class I diversity is believed to have contributed to the transmission of the tumour allograft through devil populations. Here, we report low MHC class II variability in this species, with DA β chain genes (Saha-DAB1, 2 and 3) exhibiting very limited diversity and the sole α chain gene (Saha-DAA) monomorphic. Three, six and three alleles were found at Saha-DAB1, 2 and 3, respectively, with a predominant allele found at each locus. Heterozygosity at these three loci is low in the eastern population and modestly higher in northwestern individuals. The results are indicative of a selective sweep likely due to an infectious disease resulting in the fixation of selectively favoured alleles and depletion of genetic diversity at devil class II loci. Several attempts were made to isolate the other marsupial classical class II gene family, namely, DB, resulting in only one DBB pseudogene being found. These findings further support the view that this species has a compromised capacity to respond to pathogen evolution, emerging infectious diseases and environmental changes.

Recent studies clearly demonstrate major histocompatibility complex (MHC) class I expression in the brain plays an important functional role in neural development and plasticity. A previous study from our laboratory demonstrated the temporal and spatial expression patterns of classical MHC class I molecules in the brain of C57 mice. Studies regarding non-classical MHC class I molecules remain limited. Here we examine the expression of non-classical MHC class I molecules in mouse central nervous system (CNS) during embryonic and postnatal developmental stages using in situ hybridization and immunofluorescence. We find non-classical MHC class I molecules, M3/T22/Q1, are expressed in the cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum during embryonic developmental stages. During the postnatal period from P0 to adult, non-classical MHC class I mRNAs are detected in olfactory bulb, hippocampus, cerebellum and some nerve nuclei. Overall, the expression patterns of non-classical MHC class I molecules are similar to those of classical MHC class I molecules in the developing mouse brain. In addition, non-classical MHC class I molecules are present in the H2-K(b) and H2-D(b) double knock-out mice where their expression levels are greatly increased within the same locations as compared to wild type mice. The elucidation and discovery of the expression profile of MHC class I molecules during development is important for supporting an enhanced understanding of their physiological and potential pathological roles within the CNS.

Sequence variation in a 216 bp portion of the major histocompatibility complex (MHC) II B1 domain was examined in 74 individual lake trout (Salvelinus namaycush) from different locations in Lake Superior. Forty-three alleles were obtained which encoded 71-72 amino acids of the mature protein. These sequences were compared with previous data obtained from five Pacific salmon species and Atlantic salmon using the same primers. Although all of the lake trout alleles clustered together in the neighbor-joining analysis of amino acid sequences, one amino acid allelic lineage was shared with Atlantic salmon (Salmo salar), a species in another genus which probably diverged from Salvelinus more than 10-20 million years ago. As shown previously in other salmonids, the level of nonsynonymous nucleotide substitution (d(N)) exceeded the level of synonymous substitution (d(S)). The level of nucleotide diversity at the MHC class II B1 locus was considerably higher in lake trout than in the Pacific salmon (genus Oncorhynchus). These results are consistent with the hypothesis that lake trout colonized Lake Superior from more than one refuge following the Wisconsin glaciation. Recent population bottlenecks may have reduced nucleotide diversity in Pacific salmon populations.

Study of major histocompatibility complex (MHC) loci has gained great popularity in recent years, partly due to their function in protecting vertebrates from infections. This is of particular interest in amphibians on account of major threats many species face from emergent diseases such as chytridiomycosis. In this study we compare levels of diversity in an expressedMHC class II locus with neutral genetic diversity at microsatellite loci in natterjack toad (Bufo (Epidalea) calamita) populations across the whole of the species’ biogeographical range. Variation at both classes of loci was high in the glacial refugium areas (REF) and much lower in postglacial expansion areas (PGE), especially in range edge populations. Although there was clear evidence that the MHC locus was influenced by positive selection in the past, congruence with the neutral markers suggested that historical demographic events were the main force shaping MHC variation in the PGE area. Both neutral and adaptive genetic variation declined with distance from glacial refugia. Nevertheless, there were also some indications from differential isolation by distance and allele abundance patterns that weak effects of selection have been superimposed on the main drift effect in the PGE zone. PMID:24937211

Background Ocular hypertension is a major risk factor for glaucoma, a neurodegenerative disease characterized by an irreversible decrease in ganglion cells and their axons. Macroglial and microglial cells appear to play an important role in the pathogenic mechanisms of the disease. Here, we study the effects of laser-induced ocular hypertension (OHT) in the macroglia, microglia and retinal ganglion cells (RGCs) of eyes with OHT (OHT-eyes) and contralateral eyes two weeks after lasering. Methods Two groups of adult Swiss mice were used: age-matched control (naïve, n = 9); and lasered (n = 9). In the lasered animals, both OHT-eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against glial fibrillary acid protein (GFAP), neurofilament of 200kD (NF-200), ionized calcium binding adaptor molecule (Iba-1) and major histocompatibility complex class II molecule (MHC-II). The GFAP-labeled retinal area (GFAP-RA), the intensity of GFAP immunoreaction (GFAP-IR), and the number of astrocytes and NF-200 + RGCs were quantified. Results In comparison with naïve: i) astrocytes were more robust in contralateral eyes. In OHT-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense GFAP-IR (P ii) GFAP-RA was increased in contralateral (P <0.05) and decreased in OHT-eyes (P <0.001); iii) the mean intensity of GFAP-IR was higher in OHT-eyes (P MHC-II was upregulated on macroglia and microglia. In microglia, it was similarly expressed in contralateral

The major histocompatibility complex (MHC) hosts the most polymorphic genes ever described in vertebrates. The MHC triggers the adaptive branch of the immune response, and its extraordinary variability is considered an evolutionary consequence of pathogen pressure. The last few years have witnessed the characterization of the MHC multigene family in a large diversity of bird species, unraveling important differences in its polymorphism, complexity, and evolution. Here, we characterize the first MHC class II B sequences isolated from a Rallidae species, the Eurasian Coot Fulica atra. A next-generation sequencing approach revealed up to 265 alleles that translated into 251 different amino acid sequences (β chain, exon 2) in 902 individuals. Bayesian inference identified up to 19 codons within the presumptive peptide-binding region showing pervasive evidence of positive, diversifying selection. Our analyses also detected a significant excess of high-frequency segregating sites (average Tajima's D = 2.36, P < 0.05), indicative of balancing selection. We found one to six different alleles per individual, consistent with the occurrence of at least three MHC class II B gene duplicates. However, the genotypes comprised of three alleles were by far the most abundant in the population investigated (49.4%), followed by those with two (29.6%) and four (17.5%) alleles. We suggest that these proportions are in agreement with the segregation of MHC haplotypes differing in gene copy number. The most widespread segregating haplotypes, according to our findings, would contain one single gene or two genes. The MHC class II of the Eurasian Coot is a valuable system to investigate the evolutionary implications of gene copy variation and extensive variability, the greatest ever found, to the best of our knowledge, in a wild population of a non-passerine bird. PMID:24683452

The major histocompatibility complex (MHC) hosts the most polymorphic genes ever described in vertebrates. The MHC triggers the adaptive branch of the immune response, and its extraordinary variability is considered an evolutionary consequence of pathogen pressure. The last few years have witnessed the characterization of the MHC multigene family in a large diversity of bird species, unraveling important differences in its polymorphism, complexity, and evolution. Here, we characterize the first MHC class II B sequences isolated from a Rallidae species, the Eurasian Coot Fulica atra. A next-generation sequencing approach revealed up to 265 alleles that translated into 251 different amino acid sequences (β chain, exon 2) in 902 individuals. Bayesian inference identified up to 19 codons within the presumptive peptide-binding region showing pervasive evidence of positive, diversifying selection. Our analyses also detected a significant excess of high-frequency segregating sites (average Tajima's D = 2.36, P < 0.05), indicative of balancing selection. We found one to six different alleles per individual, consistent with the occurrence of at least three MHC class II B gene duplicates. However, the genotypes comprised of three alleles were by far the most abundant in the population investigated (49.4%), followed by those with two (29.6%) and four (17.5%) alleles. We suggest that these proportions are in agreement with the segregation of MHC haplotypes differing in gene copy number. The most widespread segregating haplotypes, according to our findings, would contain one single gene or two genes. The MHC class II of the Eurasian Coot is a valuable system to investigate the evolutionary implications of gene copy variation and extensive variability, the greatest ever found, to the best of our knowledge, in a wild population of a non-passerine bird.

Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45+, MHC class II+ and CD11b+ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry+ muscle fibers was dependent on the MHCII-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity. PMID:25299691

Vaccination with naked DNA holds great promise but immunogenicity needs to be improved. DNA constructs encoding bivalent proteins that bind antigen-presenting cells (APC) for delivery of antigen have been shown to enhance T and B cell responses and protection in tumour challenge experiments. However, the mechanism for the increased potency remains to be determined. Here we have constructed DNA vaccines that express the fluorescent protein mCherry, a strategy which allowed tracking of vaccine proteins. Transfected muscle fibres in mice were visualized, and their relationship to infiltrating mononuclear cells could be determined. Interestingly, muscle fibers that produced MHC class II-specific dimeric vaccine proteins with mCherry were for weeks surrounded by a localized intense cellular infiltrate composed of CD45+, MHC class II+ and CD11b+ cells. Increasing numbers of eosinophils were observed among the infiltrating cells from day 7 after immunization. The local infiltrate surrounding mCherry+ muscle fibers was dependent on the MHCII-specificity of the vaccine proteins since the control, a non-targeted vaccine protein, failed to induce similar infiltrates. Chemokines measured on day 3 in immunized muscle indicate both a DNA effect and an electroporation effect. No influence of targeting was observed. These results contribute to our understanding for why targeted DNA vaccines have an improved immunogenicity.

In murine collagen-induced arthritis (CIA), self-reactive T cells can recognize peptide antigens derived from type-II collagen (CII). Activation of T cells is an important mediator of autoimmune diseases. Thus, T cells have become a focal point of study to treat autoimmune diseases. In this study, we evaluated the efficacy of recombinant MHC class II molecules in the regulation of antigen-specific T cells by using a self peptide derived from CII (CII260-274; IAGFKGEQGPKGEPG) linked to mouse I-A(q) in a murine CIA model. We found that recombinant I-A(q)/CII260-274 molecules could be recognized by CII-specific T cells and inhibit the same T cells in vitro. Furthermore, the development of CIA in mice was successfully prevented by in vivo injection of recombinant I-A(q)/CII260-274 molecules. Thus, treatment with recombinant soluble MHC class II molecules in complex with an immunodominant self-peptide might offer a potential therapeutic for chronic inflammation in autoimmune disease such as rheumatoid arthritis. [BMB Reports 2016; 49(6): 331-336].

The etiology of alopecia areata (AA), a putative autoimmune disease characterized by sudden hair loss, has remained obscure. It is not understood, how the characteristic inflammatory infiltrate that selectively attacks anagen hair follicles in AA is generated. We hypothesize that this reflects an unexplored form of autoimmunity, a cytotoxic T cell attack on rhythmically synthesized autoantigens normally sequestered by a lack or very low level of MHC class I (MHC I)-expression, and suggest the following mechanism of AA pathogenesis: Microtrauma, neurogenic inflammation, or microbial antigens cause a localized breakdown of MHC I-"negativity" in the proximal anagen hair bulb via proinflammatory cytokines. This exposes autoantigens derived from melanogenesis-related proteins (MRP-DP), which are only generated during anagen, and triggers two successive waves of autoimmune responses: CD8+ cytotoxic T cells initiate AA after recognizing MRP-DP abnormally presented by MHC I molecules on hair matrix melanocytes and/or keratinocytes; a secondary attack, carried by CD4+ T cells and antigen presenting cells, is then mounted against MHC class II--presented additional autoantigens exposed by damaged melanocytes and keratinocytes. The latter causes most of the follicular damage, and extrafollicular disease, and depends greatly on the immunogenetic background of affected individuals. This unifying hypothesis explains the clinical heterogeneity and all salient features of AA, and argues that only the unlikely coincidence of multiple predisposing events triggers AA. The suppression of MHC I--expression and synthesis of MRP in the hair bulb, and the "tolerization" of MRP-DP autoreactive CD8+ T cells may be promising strategies for treating AA. PMID:7716973

Recombination, demographic history, drift and selection influence the extent of linkage disequilibrium (LD) in the human genome, but their relative contributions remain unclear. To investigate the effect of meiotic recombination versus population history on LD, three populations with different demographic histories (UK north Europeans, Saami and Zimbabweans) were genotyped for high-frequency single-nucleotide polymorphisms (SNPs) across a 75 kb DNA segment of the MHC class II region. This region spans three well-characterized recombination hotspots and a 60 kb long LD block. Despite a high level of underlying haplotype diversity and considerable divergence in haplotype composition between populations, all three populations showed very similar patterns of LD. Surprisingly, the entire 60 kb LD block was present even in Africans, although it was relatively difficult to detect owing to a systematic deficiency of high frequency SNPs. In contrast, DNA within recombination hotspots did not show this low nucleotide diversity in Africans. Thus, while population history has some influence on LD, our findings suggest that recombination hotspots play a major global role in shaping LD patterns as well as helping to maintain localized SNP diversity in this region of the MHC.

The diversity of the MHC class II region in non-human primates is a focus of biomedical research because this region plays a crucial role in the recognition of antigens in the immune system. In particular, the chimpanzee [Pan troglodytes (Patr)], which belongs to the superfamily Hominoidea, has been used as a human model for the study of diseases such as human hepatitis C virus (HCV), human hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, to which only humans and chimpanzees are susceptible. In the present study, polymorphisms of the MHC-DPB1 gene (Patr-DPB1) in a chimpanzee colony in Japan were examined using a stepwise polymerase chain reaction (PCR) technique. In order to design a suitable primer pair which would amplify exon 2 of the Patr-DPB1 gene, a fragment of approximately 8 kb from exon 1 to exon 3 was amplified from chimpanzee genomic DNA. After designing a 500-bp primer pair at the 3' region of intron 1 and the 5' region of intron 2, analysis of DPB1 exon 2 alleles of each chimpanzee was carried out. Twenty-two chimpanzees were used in our study, and we identified seven alleles by sequence analysis on the Patr-DPB1 gene, including one new allele. The obtained nucleotide sequence patterns suggest that Patr-DPB1 alleles emerge by genetic variations such as the exchange of sequence motifs and the accumulation of point mutations.

Previous studies have shown that homozygous mutations between the LMP2 and DNA loci in the human MHC cause class II molecules to be abnormally conformed and unstable in the presence of SDS at low temperature, and impede class II-associated Ag processing and presentation. These abnormalities result from impaired ability to form intracellular class II/peptide complexes that predominate in normal cells. We show in this work that this defect results from deficient expression of either the DMA or the DMB gene. Human B-LCL.174 (DR3) cells, which have a deletion of all known expressible genes in the class II region, express transgene-encoded HLA-DR3, but have the abnormalities. Transfer of cosmid HA14, which contains the DMA and DMB genes, into .174 (DR3) cells restored normal DR3 conformation, stability in 0.4% SDS at 0{degrees}, and ability to process and present tetanus toxoid, but only when both DMA and DMB mRNAs were present. The requirement for both genetic expressions in engendering normal phenotypes was confirmed by transferring the cloned genes into .174 (DR3) cells separately or together. Because normal phenotypes were fully restored in transferent cells expressing DMA plus DMB, other genes in the {approximately} 1-mb homozygous class II region deletion in .174 (DR3) cells either do not participate in or are dispensable for apparently normal production of intracellular class II/peptide complexes. The properties of DM-deficient EBV-transformed B lymphoblastoid cell lines (LCLs) suggest ways of identifying humans in whom DM deficiency contributes to congenital immunodeficiency and malignancy. 67 refs., 5 figs., 1 tab.

Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001 PMID:25860507

In the present study, exon 2 of major histocompatibility complex (MHC) class II DQB gene from 39 gayals (Bos frontalis) was isolated, characterized and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 36 DQB exon 2 variants among 39 gayals. These variants exhibited a high degree of nucleotide and amino acid substitutions with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (dS) and non-synonymous (dN) substitution. The gayals were observed to be under strong balancing selection in the DQB exon 2 PBS (dN = 0.094, P = 0.001). It appears that this variability among gayals could confer the ability to mount immune responses to a wide variety of peptides or pathogens.

In the present study, exon 2 of major histocompatibility complex (MHC) class II DQB gene from 39 gayals (Bos frontalis) was isolated, characterized and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 36 DQB exon 2 variants among 39 gayals. These variants exhibited a high degree of nucleotide and amino acid substitutions with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (d S) and non-synonymous (d N) substitution. The gayals were observed to be under strong balancing selection in the DQB exon 2 PBS (d N = 0.094, P = 0.001). It appears that this variability among gayals could confer the ability to mount immune responses to a wide variety of peptides or pathogens. PMID:26019566

There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

The major histocompatibility complex (MHC) comprises many genes, some of which are polymorphic with numerous alleles. Sequence variation among alleles is most pronounced in exon 2 of the class II genes, which encodes the α1 and β1 domains that form the antigen-binding site (ABS) for the presentation of peptides. The MHC thus plays an important role in pathogen defense. European badgers (Meles meles) are a good species in which to study the MHC, as they harbor a variety of pathogens. We present the first characterization of MHC class II genes, isolated from genomic DNA (gDNA) and complementary DNA (cDNA), in the European badger. Examination of seven individuals revealed four DRB, two DQB, two DQA, and two DRA putatively functional gDNA sequences. All of these sequences, except DRA, exhibited high variability in exon 2; DRB had the highest variability. The ABS codons demonstrated high variability, due potentially to balancing selection, while non-ABS codons had lower variability. Positively selected sites were detected in DRB and DQA. Phylogenetic analysis demonstrated trans-species polymorphism of class II genes. Comparison with cDNA from whole blood revealed that only DRB had a transcription pattern reflecting the alleles that were present in the gDNA, while the other three genes had disparities between gDNA and cDNA. Only one sequence was transcribed, even though two gDNA sequences were present, from each of both DQB and DRA. Our characterization of badger MHC sequences forms a basis for further studies of MHC variability, mate choice, and pathogen resistance in this, and other, species.

Cells use multiple autophagy pathways to sequester macromolecules, senescent organelles, and pathogens. Several conserved isoforms of the lysosome-associated membrane protein-2 (LAMP-2) regulate these pathways influencing immune recognition and responses. LAMP-2A is required for chaperone-mediated autophagy (CMA), which promotes Ag capture and MHC class II (MHCII) presentation in B cells and signaling in T cells. LAMP-2B regulates lysosome maturation to impact macroautophagy and phagocytosis. Yet, far less is known about LAMP-2C function. Whereas LAMP2A and LAMP2B mRNA were broadly detected in human tissues, LAMP2C expression was more limited. Transcripts for the three LAMP2 isoforms increased with B cell activation, although specific gene induction varied depending on TLR versus BCR engagement. To examine LAMP-2C function in human B cells and specifically its role in Ag presentation, we used ectopic gene expression. Increased LAMP-2C expression in B cells did not alter MHCII expression or invariant chain processing, but did perturb cytoplasmic Ag presentation via CMA. MHCII presentation of epitopes from exogenous and membrane Ags was not affected by LAMP-2C expression in B cells. Similarly, changes in B cell LAMP-2C expression did not impact macroautophagy. The gene expression of other LAMP2 isoforms and proteasome and lysosomal proteases activities were unperturbed by LAMP-2C ectopic expression. LAMP-2C levels modulated the steady-state expression of several cytoplasmic proteins that are targeted for degradation by CMA and diminished peptide translocation via this pathway. Thus, LAMP-2C serves as a natural inhibitor of CMA that can selectively skew MHCII presentation of cytoplasmic Ags.

Cannabinoid receptors are known to be expressed in microglia; however, their involvement in specific aspects of microglial immune function has not been demonstrated. Many effects of cannabinoids are mediated by two G-protein coupled receptors, designated CB1 and CB2. We have shown that the CB1 receptor is expressed in microglia that also expressMHC class II antigen (J. Neuroimmunol. 82 (1998) 13-21). In our present study, we have analyzed the effect of cannabinoid agonist CP55,940 on MHC class IIexpression on the surface of IFN-gamma induced microglial cells by flow cytometry. CP55,940 blocked the class IIMHCexpression induced by IFN-gamma. It has been shown that the regulation of class IIMHC genes occurs primarily at the transcriptional level, and a non-DNA binding protein, class II transactivator (CIITA), has been shown to be the master activator for class II transcription. We find that mRNA levels of CIITA are increased in IFN-gamma induced EOC 20 microglial cells and that this increase is almost entirely eliminated by the cannabinoid agonist CP55,940. These data suggests that cannabinoids affect MHC class IIexpression through actions on CIITA at the transcriptional level.

Class II genes of the bovine major histocompatibility complex (MHC) have been investigated by Southern blot analysis using human DNA probes. Previous studies revealed the presence of bovine DO beta, DQ alpha, DQ beta, DR alpha, and DR beta genes, and restriction fragment length polymorphisms for each of these genes were documented. In the present study, the presence of three additional class II genes, designated DZ alpha, DY alpha, and DY beta, are reported. DZ alpha was assumed to correspond to the human DZ alpha gene while the other two were designated DY because their relationship to human class II genes could not be firmly established. The linkage relationships among bovine class II genes and two additional loci, TCP1B and C4, were investigated by family segregation analysis and analysis of linkage disequilibrium. The results clearly indicated that all these loci belong to the same linkage group. This linkage group is divided into two subregions separated by a fairly high recombination frequency. One region includes the C4, DQ alpha, DQ beta, DR alpha, and DR beta loci and the other one is composed of the DO beta, DY alpha, DY beta, and TCP1B loci. No recombinant was observed within any of these subregions and there was a strong or fairly strong linkage disequilibrium between loci within groups. In contrast, as many as five recombinants among three different families were detected in the interval between these subregions giving a recombination frequency estimate of 0.17 +/- 0.07. The fairly high recombination frequency observed between class II genes in cattle is strikingly different from the corresponding recombination estimates in man and mouse. The finding implies either a much larger molecular distance between some of the bovine class II genes or alternatively the presence of a recombinational "hot spot" in the bovine class II region.

Background Previously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class IIMHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activation. Methodology/Principal Findings MΦ of CBA/j mice were infected with Leishmania donovani (I-MΦ). Two different anti-Aκ mAbs were used to monitor the status of MHC-II protein under parasitized condition. One of them (11.5–2) was conformation specific, whereas the other one (10.2.16) was not. Under parasitized condition, the binding of 11.5–2 decreased significantly with respect to the normal counterpart, whereas that of 10.2.16 remained unaltered. The binding of 11.5–2 was restored to normal upon liposomal delivery of cholesterol in I-MΦ. By molecular dynamics (MD) simulation studies we found that there was considerable conformational fluctuation in the transmembrane domain of the MHC-II protein in the presence of membrane cholesterol than in its absence, which possibly influenced the distal peptide binding groove. This was evident from the faster dissociation of the cognate peptide from peptide-MHC complex under parasitized condition, which could be corrected by liposomal delivery of cholesterol in I-MΦ. Conclusion The decrease in membrane cholesterol in I-MΦ may lead to altered conformation of MHCII, and this may contribute to a faster dissociation of

Antigenic peptides or cancer peptide vaccines can be directly delivered to cancer patients to produce immunologic responses against cancer cells. Specifically, designed peptides can associate with Major Histocompatibility Complex (MHC) class I or II molecules on the cell surface of antigen presenting cells activating anti-tumor effector mechanisms by triggering helper T cell (Th) or cytotoxic T cells (CTL). In general, high binding to MHCs approximately correlates with in vivo immunogenicity. Consequently, a molecular docking technique was run on a library of novel discontinuous peptides predicted by PEPOP from Human epidermal growth factor receptor 2 (HER2 ECD) subdomain III. This technique is expected to improve the prediction accuracy in order to identify the best MHC class I and II binder peptides. Molecular docking analysis through GOLD identified the peptide 1412 as the best MHC binder peptide to both MHC class I and II molecules used in the study. The GOLD results predicted HLA-DR4, HLA-DP2 and TCR as the most often targeted receptors by the peptide 1412. These findings, based on bioinformatics analyses, can be exploited in further experimental analyses in vaccine design and cancer therapy to find possible proper approaches providing beneficial effects.

The development of standardised nomenclatures with associated databases containing reference sequences for alleles at polymorphic loci within the Major Histocompatibility Complex (MHC) has been facilitated by the development of the Immuno Polymorphism Database (IPD-MHC). Recently, included within I...

Interleukin (IL)-17 expressing CD4+ T lymphocytes (Th17 cells) naturally reside in the intestine where specific cytokines and microbiota, such as segmented filamentous bacteria (SFB), promote their differentiation. Intestinal Th17 cells are believed to initially differentiate in the GALT and/or mesenteric lymph nodes (mLN) upon antigen encounter and subsequently home to the lamina propria (LP) where they mediate effector functions. However, whether GALT and/or mLN are required for intestinal Th17 differentiation, and how microbiota containing SFB regulate antigen-specific intestinal Th17 cells remain poorly defined. Here we observed that naïve CD4+ T cells were abundant in the intestinal LP prior to weaning and that the accumulation of Th17 cells in response to microbiota containing SFB occurred in the absence of lymphotoxin (LT)-dependent lymphoid structures and the spleen. Furthermore, the differentiation of intestinal Th17 cells in the presence of microbiota containing SFB was dependent on MHCIIexpression by CD11c+ cells. Lastly, the differentiation of antigen-specific Th17 cells required both the presence of cognate antigen and microbiota containing SFB. These findings suggest that microbiota containing SFB create an intestinal milieu that may induce antigen-specific Th17 differentiation against food and/or bacterial antigens directly in the intestinal LP. PMID:24899505

The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated "HURRAH" based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ∼ DRB1 ∼ DRB3 ∼ DQA1 ∼ DQB2 (H1) and DRA1*02 ∼ DRB2 ∼ DRB4 ∼ DQA2 ∼ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens.

The major histocompatibility complex (MHC) plays an important role in immune response. Avian MHCs are not well characterized, only reporting highly compact Galliformes MHCs and extensively fragmented zebra finch MHC. We report the first genomic structure of an endangered Pelecaniformes (crested ibis) MHC containing 54 genes in three regions spanning ~500 kb. In contrast to the loose BG (26 loci within 265 kb) and Class I (11 within 150) genomic structures, the Core Region is condensed (17 within 85). Furthermore, this Region exhibits a COL11A2 gene, followed by four tandem MHC class II αβ dyads retaining two suites of anciently duplicated “αβ” lineages. Thus, the crested ibis MHC structure is entirely different from the known avian MHC architectures but similar to that of mammalian MHCs, suggesting that the fundamental structure of ancestral avian class II MHCs should be “COL11A2-IIαβ1-IIαβ2.” The gene structures, residue characteristics, and expression levels of the five class I genes reveal inter-locus functional divergence. However, phylogenetic analysis indicates that these five genes generate a well-supported intra-species clade, showing evidence for recent duplications. Our analyses suggest dramatic structural variation among avian MHC lineages, help elucidate avian MHC evolution, and provide a foundation for future conservation studies. PMID:25608659

The major histocompatibility complex (MHC) is integral to the vertebrate adaptive immune system. Characterizing diversity at functional MHC genes is invaluable for elucidating patterns of adaptive variation in wild populations, and is particularly interesting in species of conservation concern, which may suffer from reduced genetic diversity and compromised disease resilience. Here, we use next generation sequencing to investigate MHC class II B (MHCIIB) diversity in two sister taxa of New Zealand birds: South Island saddleback (SIS), Philesturnus carunculatus, and North Island saddleback (NIS), Philesturnus rufusater. These two species represent a passerine family outside the more extensively studied Passerida infraorder, and both have experienced historic bottlenecks. We examined exon 2 sequence data from populations that represent the majority of genetic diversity remaining in each species. A high level of locus co-amplification was detected, with from 1 to 4 and 3 to 12 putative alleles per individual for South and North Island birds, respectively. We found strong evidence for historic balancing selection in peptide-binding regions of putative alleles, and we identified a cluster combining non-classical loci and pseudogene sequences from both species, although no sequences were shared between the species. Fewer total alleles and fewer alleles per bird in SIS may be a consequence of their more severe bottleneck history; however, overall nucleotide diversity was similar between the species. Our characterization of MHCIIB diversity in two closely related species of New Zealand saddlebacks provides an important step in understanding the mechanisms shaping MHC diversity in wild, bottlenecked populations.

The genetic diversity of MHC class II DQ genes was investigated in riverine buffalo (Bubalus bubalis) by PCR-RFLP and sequencing. Highly variable regions (exons 2-3) of DQ genes were amplified from 152 buffaloes and genotyped by PCR-RFLP. Alleles identified by differential restriction patterns were sequenced for the characterization. PCR-RFLP was a rapid method to discriminate between DQA1 and duplicated DQA2 genes in buffalo, however, the method appeared to be inadequate for determining the more complicated DQB genotypes. A total of 7 and 10 alleles were identified for DQA and DQB loci, respectively. Nucleotide as well as amino acid variations among DQ alleles particularly at peptide binding regions were high. Such variations were as expected higher in DQB than DQA alleles. The phylogenetic analysis for both genes revealed the grouping of alleles into two major sub-groups with higher genetic divergence. High divergence among DQ allelic families and the isolation of two diverse DQA and DQB sequences from individual samples indicated duplication of DQ loci was similar in buffalo to other ruminants.

Single-molecule epifluorescence microscopy was used to observe the translational motion of GPI-linked and native I-E(k) class IIMHC membrane proteins in the plasma membrane of CHO cells. The purpose of the study was to look for deviations from Brownian diffusion that might arise from barriers to this motion. Detergent extraction had suggested that these proteins may be confined to lipid microdomains in the plasma membrane. The individual I-E(k) proteins were visualized with a Cy5-labeled peptide that binds to a specific extracytoplasmic site common to both proteins. Single-molecule trajectories were used to compute a radial distribution of displacements, yielding average diffusion coefficients equal to 0.22 (GPI-linked I-E(k)) and 0.18 microm(2)/s (native I-E(k)). The relative diffusion of pairs of proteins was also studied for intermolecular separations in the range 0.3-1.0 microm, to distinguish between free diffusion of a protein molecule and diffusion of proteins restricted to a rapidly diffusing small domain. Both analyses show that motion is predominantly Brownian. This study finds no strong evidence for significant confinement of either GPI-linked or native I-E(k) in the plasma membrane of CHO cells. PMID:12414700

Background Zebrafish (Danio rerio) is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry. In this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput RNA sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with Edwardsiella tarda live attenuated vaccine. Results Average reads of 18.13 million and 14.27 million were obtained from livers of zebrafish immunized with phosphate buffered saline (mock) and E. tarda vaccine (WED), respectively. The reads were annotated with the Ensembl zebrafish database before differential expressed genes sequencing (DESeq) comparative analysis, which identified 4565 significantly differentially expressed genes (2186 up-regulated and 2379 down-regulated in WED; p<0.05). Among those, functional classifications were found in the Gene Ontology database for 3891 and in the Kyoto Encyclopedia of Genes and Genomes database for 3467. Several pathways involved in acute phase response, complement activation, immune/defense response, and antigen processing and presentation were remarkably affected at the early stage of WED immunization. Further qPCR analysis confirmed that the genes encoding the factors involved in major histocompatibility complex (MHC)-I processing pathway were up-regulated, while those involved in MHC-II pathway were down-regulated. Conclusion These data provided insights into the molecular mechanisms underlying zebrafish immune response to WED immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts. PMID:22805612

It is commonly thought that autoimmune diseases are caused by the breakdown of self-tolerance, which suggests the recognition of specific antigens by autoreactive CD4+ T cells contribute to the specificity of autoimmune diseases (Marrack et al., 2001; Mathis and Benoist, 2004). In several cases, however, even for diseases associated with class II major histocompatibility complex (MHC) alleles, the causative tissue-specific antigens recognized by memory/activated CD4+ T cells have not been established (Mocci et al., 2000; Skapenko et al., 2005). Rheumatoid arthritis (RA) and arthritis in F759 knock-in mice (F759 mice) are such examples (Atsumi et al., 2002; Brennan et al., 2002; Falgarone et al., 2009). These include associations with class IIMHC and CD4 molecules; increased numbers of memory/activated CD4+ T cells; and improved outcomes in response to suppressions and/or deficiencies in class IIMHC molecules, CD4+ T cells, and the T cell survival cytokine IL-7. Regarding the development of arthritis in F759 mice, it is not only the immune system, but also non-immune tissue that are involved, indicating that the importance of their interactions (Sawa et al., 2006, 2009; Ogura et al., 2008; Hirano, 2010; Murakami et al., 2011). Furthermore, we have shown that local events such as microbleeding together with an accumulation of activated CD4+ T cells in a manner independent of tissue antigen-recognitions induces arthritis in the joints of F759 mice (Murakami et al., 2011). For example, local microbleeding-mediated CCL20 expression induce such an accumulation, causing arthritis development via chronic activation of an IL-17A-dependent IL-6 signaling amplification loop in type 1 collagen+ cells that is triggered by CD4+ T cell-derived cytokine(s) such as IL-17A, which leads to the synergistic activation of STAT3 and NFκB in non-hematopoietic cells in the joint (Murakami et al., 2011). We named this loop the IL-6-mediated inflammation amplifier, or IL-6 amplifier for

Peptide presentation by the major histocompatibility complex (MHC) is a key process for triggering a specific immune response. Studying peptide-MHC (pMHC) binding from a structural-based approach has potential for reducing the costs of investigation into vaccine development. This study involved using two semi-empirical quantum chemistry methods (PM7 and FMO-DFTB) for computing the binding energies of peptides bonded to HLA-DR1 and HLA-DR2. We found that key stabilising water molecules involved in the peptide binding mechanism were required for finding high correlation with IC50 experimental values. Our proposal is computationally non-intensive, and is a reliable alternative for studying pMHC binding interactions.

The cane toad has gained notoriety for its invasion across the Australian landscape, with significant impacts on the native Australian fauna. The invasion has accelerated over time, with invading cane toads adapted for highly dispersive traits. This, however, has come at the cost of the immune system, with lower investment in some immune functions. To investigate the cane toad's immunogenetics, we characterized four major histocompatibility complex (MHC) class IIA and three MHC class IIB loci. Preliminary observations suggest very low allelic diversity at all loci. We also observed various splice isoforms. One isoform seen at one class IIA and two class IIB loci was missing exon 2, which is essential to peptide binding and presentation. The other isoform, observed at a class IIA locus, is likely to be a soluble MHC product. These results may suggest a significant role of alternative splicing of MHC loci in the Australian cane toad.

HLA-DM is required for efficient peptide exchange on class IIMHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class IIMHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHCII protein in the region of the α-subunit 310 helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange. PMID:22084083

HLA-DM is required for efficient peptide exchange on class IIMHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class IIMHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHCII protein in the region of the α-subunit 3(10) helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.

While the anuran Xenopus comprises one of the best characterized nonmammalian taxa regarding the major histocompatibility complex (MHC), the organization of this gene complex has never been studied in other anurans, and information on amphibian MHC (other than Xenopus) is generally very scarce. Here, we describe the characterization of the first MHC class II B cDNA sequences from a nonmodel anuran species, the European fire-bellied toad (Bombina bombina). We isolated two transcript sequences differing substantially in amino acid composition and length within the beta2 domain. To investigate the variability of the peptide binding region in this species, we sequenced a 158-bp large fragment from wild B. bombina (n = 20) and identified eight distinct alleles. All substitutions but one were nonsynonymous, and many of the highly polymorphic sites corresponded with amino acid positions known to be involved in antigen binding. The level of variation we found in B. bombina was similar compared to that previously found in a comparable sample of a wild urodelan species, Ambystoma tigrinum, and to that found in Xenopus laevis. Based on the cDNA data and the individual's allelic diversity, we conclude that Bombina possesses at least two class II B loci. With our new beta1 primers, we were able to generate sequences in other species of anurans. We provide here a first phylogenetic analysis of this gene in amphibians.

The polymorphic major histocompatibility complex (MHC) has gained a specific relevance in pathogen resistance and mate choice. Particularly the antigen-binding site (ABS), encoded by exon 2 of the DRB class II gene, exhibits numerous alleles and extensive sequence variations between alleles. A lack of MHC variability has attributed to instances such as bottleneck effects or relaxed selection pressure and has a certain impact on the long-term viability of the species concerned. As a result of seriously decreased population density during the last century, the current population of the endangered European mink (Mustela lutreola, L. 1761) has suffered from geographic isolation. In this study, we amplified a partial sequence of the MHC class II DRB exon 2 (229 bp), assessed the degree of genetic variation and compared the variability with those of other Mustelidae. As a result, nine alleles were detected in 20 investigated individuals, which differ from each other by four to 25 nucleotide substitutions (two to 11 amino acid substitutions). Whilst an equal ratio for synonymous and non-synonymous substitutions was found inside the ABS, synonymous substitutions were significantly higher than non-synonymous substitutions in the non-ABS region. Results might indicate that no positive selection exists within the ex situ population of M. lutreola, at least in the analysed fragment. In addition, phylogenetic analyses support the trans-species model of evolution.

Although it is generally believed that CD4(+) T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHCII-restricted CD3(+)CD4(-)CD8(-) (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.

Holstein cattle dominate the global milk production industry because of their outstanding milk production, however, this breed is susceptible to tropical endemic pathogens and suffers from heat stress and thus fewer Holstein populations are raised in tropical areas. The bovine major histocompatibility complex (BoLA)-DRB3 class II gene is used as a marker for disease and immunological traits, and its polymorphism has been studied extensively in Holstein cattle from temperate and cold regions. We studied the genetic diversity of the BoLA-DRB3 gene in South American Holstein populations to determine whether tropical populations have diverged from those bred in temperate and cold regions by selection and/or crossbreeding with local native breeds. We specifically studied Exon 2 of this gene from 855 South American Holstein individuals by a polymerase chain reaction (PCR) sequence-based typing method. We found a high degree of gene diversity at the allelic (Na > 20 and He > 0.87) and molecular (π > 0.080) levels, but a low degree of population structure (FST = 0.009215). A principal components analysis and tree showed that the Bolivian subtropical population had the largest genetic divergence compared with Holsteins bred in temperate or cold regions, and that this population was closely related to Bolivian Creole cattle. Our results suggest that Holstein genetic divergence can be explained by selection and/or gene introgression from local germplasms. This is the first examination of BoLA-DRB3 in Holsteins adapted to tropical environments, and contributes to an ongoing effort to catalog bovine MHC allele frequencies by breed and location.

Mouse mammary tumor virus superantigens (vSAGs) are notorious for defying structural characterization, and a consensus has yet to be reached regarding their ability to bridge the TCR to MHC class II (MHCII). In this study, we determined the topology of the T cell signaling complex by examining the respective relation of vSAG7 with the MHCII molecule, MHCII-associated peptide, and TCR. We used covalently linked peptide/MHCII complexes to demonstrate that vSAG presentation is tolerant to variation in the protruding side chains of the peptide, but can be sensitive to the nature of the protruding N-terminal extension. An original approach in which vSAG was covalently linked to either MHCII chain confirmed that vSAG binds outside the peptide binding groove. Also, whereas the C-terminal vSAG segment binds to the MHCII α-chain in a conformation-sensitive manner, the membrane-proximal N-terminal domain binds the β-chain. Because both moieties of the mature vSAG remain noncovalently associated after processing, our results suggest that vSAG crosslinks MHCII molecules. Comparing different T cell hybridomas, we identified key residues on the MHCII α-chain that are differentially recognized by the CDR3β when engaged by vSAG. Finally, we show that the highly conserved tyrosine residue found in the vSAg TGXY motif is required for T cell activation. Our results reveal a novel SAG/MHCII/TCR architecture in which vSAGs coerce a near-canonical docking between MHCII and TCR that allows eschewing of traditional CDR3 binding with the associated peptide in favor of MHCII α-chain binding. Our findings highlight the plasticity of the TCR CDRs.

Major histocompatibility complex (Mhc) class II Ab genes have evolved into three distinct lineages. While lineage 2 alleles differ from lineage 1 alleles by the insertion of a retroposon in intron 2, the basis for the extremely large intron 2 in lineage 3 alleles has heretofore been undetermined. In this report, we demonstrate by nucleotide sequencing that the genomic sequences of prototypic alleles from all three lineages diverge significantly and that lineage 3 is derived from lineage 2 by two insertional events in intron 2. One insert, composed of a member of B1 short interspersed repetitive elements (SINEs), occurs 508 base pairs (bp) 3' of exon 2, and the other, 1141 bp 3' of exon 2 within the retroposon that distinguishes lineage 2 from lineage 1. To assess the evolutionary stability of these lineages and the extent of ancestral polymorphisms of Ab within Mus species, we extended our restriction site polymorphism analysis to include 86 alleles from 120 independently derived H2 haplotypes from 12 separate species and subspecies of Mus. A phylogenetic tree revealing the relationships of these Ab alleles with respect to restriction site polymorphisms, but excluding the retroposon insertions, demonstrated that these lineages have distinctive genomic structures beyond the retroposon polymorphisms. In summary, these mouse Ab genes were produced from successive retroposon insertion events. Lineage 1 and 2 were detected in a variety of Mus species, including Mus caroli, indicating that these lineages diverged more than 2 million years ago. Lineage 3 alleles were found only in the Mus musculus subspecies, suggesting that it diverged from lineage 2 more recently. These results indicate that all three lineages of Ab have persisted through several speciation events in the genus Mus.

In order to construct prokaryotic expression system of MHC classI chain-related gene A (mica) and purify MICA protein, RNAs were extracted from the peripheral blood samples and mica cDNA fragments were obtained by RT-PCR method. The cDNA for mica was ligated with cloning vector by TOPO method. The recombinant cloning vector and prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct pET-28a-MICA recombinant expression vector, then the pET-28a-MICA vector was transformed and expressed in E. coli BL21 DE3. The recombinant protein was purified by Ni-NTA Spin method. The results showed that the recombinant MICA protein expressed with soluble form in host with pET-28a-MICA vector after IPTG induction. The recombinant target protein was obtained by Ni-NTA spin purification. In conclusion, this study has constructed prokaryotic expression system of mica gene and has purified MICA protein which would help to explore the interaction between MICA and transplantation immunology.

Antigenic T cell stimulation requires interaction between the TCR of the T cell and cognate peptide-MHC molecules presented by the APC. Although studies with TCR-specific Abs and soluble peptide-MHC ligands have shown that the TCR needs to be crosslinked by two or more ligands to induce T cell stimulation, it is not understood how several MHC molecules loaded with the cognate antigenic peptide can produce crosslinking under physiological conditions. We show at the molecular level that large clusters of cognate peptide-MHC are formed at the surface of murine professional and nonprofessional APCs upon virus infection and that these clusters impinge on the stimulatory capacity of the APC. These clusters are formed by tight apposition of cognate peptide-MHC complexes in a configuration that is compatible with simultaneous engagement of two or more TCRs. This suggests that physiological expression of Ag allows formation of multivalent ligands for the TCR that permit TCR crosslinking and T cell activation.

Within each individual, a highly diverse T cell receptor (TCR) repertoire interacts with peptides presented by major histocompatibility complex (MHC) molecules. Despite extensive research, it remains controversial whether germline-encoded TCR-MHC contacts promote TCR-MHC specificity and if so, whether there exist differences in TCR V-gene compatibilities with different MHC alleles. We applied eQTL mapping to test for associations between genetic variation and TCR V-gene usage in a large human cohort. We report strong trans associations between variation in the MHC locus and TCR V-gene usage. Fine mapping of the association signals reveals specific amino acids in MHC genes that bias V-gene usage, many of which contact or are spatially proximal to the TCR or peptide. Hence, these MHC variants, several of which are linked to autoimmune diseases, can directly affect TCR-MHC interaction. These results provide the first examples of trans-QTLs mediated by protein-protein interactions, and are consistent with intrinsic TCR-MHC specificity. PMID:27479906

The bare lymphocyte syndrome is a rare combined immunodeficiency disorder associated with the absence of class I and/or class II major histocompatibility (MHC) antigens. Although it has been inferred that the immune deficiency is a consequence of disordered MHC-restricted interactions among otherwise normal cells, the biological capabilities and differentiation of B lymphocytes deficient in class IIMHC antigens have not been rigorously analyzed. We have examined the phenotypic and functional attributes of B cells with absent class IIMHC antigens. Our data demonstrate that these B cells are intrinsically defective in their responses to membrane-mediated activation stimuli. In addition, virtually all the B cells had phenotypic evidence of arrested differentiation at an immature stage. Finally, these B cells also failed to express the C3d-EBV receptor normally present on all B lymphocytes. These data indicate that class IIMHC molecules are vital participants in early events of the B cell activation cascade, and that other non-MHC membrane molecules may also be absent as a consequence of either arrested differentiation or as a result of the basic defect affecting the expression of MHC membrane antigens. PMID:3257764

We present a new method to explore interactions between peptides and major histocompatibility complex (MHC) molecules using the resultant vector of the three principal multipole terms of the electrostatic field expansion. Being that molecular interactions are driven by electrostatic interactions, we applied quantum chemistry methods to better understand variations in the electrostatic field of the MHC Class II HLA-DRbeta1*0101-HA complex. Multipole terms were studied, finding strong alterations of the field in Pocket 1 of this MHC molecule, and weak variations in other pockets, with Pocket 1>Pocket 4>Pocket 9 approximately Pocket 7>Pocket 6. Variations produced by "ideal" amino acids and by other occupying amino acids were compared. Two types of interactions were found in all pockets: a strong unspecific one (global interaction) and a weak specific interaction (differential interaction). Interactions in Pocket 1, the dominant pocket for this allele, are driven mainly by the quadrupole term, confirming the idea that aromatic rings are important in these interactions. Multipolar analysis is in agreement with experimental results, suggesting quantum chemistry methods as an adequate methodology to understand these interactions.

Major histocompatibility complex (MHC) genes are the most polymorphic genes in vertebrates and encode molecules that play a crucial role in pathogen resistance. As a result of their diversity, they have received much attention in the fields of evolutionary and conservation biology. Here, we described the genetic variation of MHC class II B (MHCIIB) exon 2 in a wild population of Hume's pheasant (Syrmaticus humiae), which has suffered a dramatic decline in population over the last three decades across its ranges in the face of heavy exploitation and habitat loss. Twenty-four distinct alleles were found in 73 S. humiae specimens. We found seven shared alleles among four geographical groups as well as six rare MHCIIB alleles. Most individuals displayed between one to five alleles, suggesting that there are at least three MHCIIB loci of the Hume's pheasant. The dN ⁄ dS ratio at putative antigen-binding sites (ABS) was significantly greater than one, indicating balancing selection is acting on MHCIIB exon 2. Additionally, recombination and gene conversion contributed to generating MHCIIB diversity in the Hume's pheasant. One to three recombination events and seventy-five significant gene conversion events were observed within the Hume's pheasant MHCIIB loci. The phylogenetic tree and network analysis revealed that the Hume's pheasant alleles do not cluster together, but are scattered through the tree or network indicating a trans-species evolutionary mode. These findings revealed the evolution of the Hume's pheasant MHC after suffering extreme habitat fragmentation.

The organization of the major histocompatibility complex (MHC) class II region in cattle was investigated by Southern blot analysis using human probes corresponding to DO, DP, DQ, and DR genes. Exon-specific probes were also employed to facilitate the assessment of the number of different bovine class II genes. The results indicated the presence of single DO beta and DR alpha genes, at least three DR beta genes, while the number of DQ genes was found to vary between MHC haplotypes. Four DQ haplotypes, DQ alpha 1 beta 1 to DQ alpha 2 beta 4, possessed a single DQ alpha and a single DQ beta gene whereas both these genes were duplicated in eight other haplotypes, DQ alpha 3 beta 5 to DQ alpha 9 beta 12. No firm evidence for the presence of bovine DP genes was obtained. The same human probes were also used to investigate the genetic polymorphism of bovine class II genes. DQ alpha, DQ beta, DR alpha, DR beta, and DO beta restriction fragment length polymorphisms (RFLPs) were resolved and in particular the DQ restriction fragment patterns were highly polymorphic. Comparison of the present result with the current knowledge of the class II region in other mammalian species suggested that the DO, DP, DQ, DR, and DZ subdivision of the class II region was established already in the ancestor of mammals. The DP genes appear to be the least conserved class II genes among mammalian species and may have been lost in cattle. The degree of polymorphism of different class II genes, as revealed by RFLP analyses, shows striking similarities between species.

Structural studies of complexes of T cell receptor (TCR) and peptide-major histocompatibility complex (MHC) have focused on TCRs specific for foreign antigens or native self. An unexplored category of TCRs includes those specific for self determinants bearing alterations resulting from disease, notably cancer. We determined here the structure of a human melanoma-specific TCR (E8) bound to the MHC molecule HLA-DR1 and an epitope from mutant triosephosphate isomerase. The structure had features intermediate between 'anti-foreign' and autoimmune TCR-peptide-MHC class II complexes that may reflect the hybrid nature of altered self. E8 manifested very low affinity for mutant triosephosphate isomerase-HLA-DR1 despite the highly tumor-reactive properties of E8 cells. A second TCR (G4) had even lower affinity but underwent peptide-specific formation of dimers, suggesting this as a mechanism for enhancing low-affinity TCR-peptide-MHC interactions for T cell activation.

Major histocompatibility complex (MHC) class II molecules play a key role in the adaptive immune system of vertebrates. Class II B genes appear to evolve in a very different manner in mammals and birds. Orthology is commonly observed among mammal loci, while genes tend to cluster phylogenetically within bird species. Here we present class II B data from a representative of another major group of amniotes, the squamates (i.e. lizards, snakes, amphisbaenians), with the ultimate goal of placing mammalian and avian MHC evolution into a broader context. In this study, eight class II B cDNA sequences were obtained from the Galápagos marine iguana (Amblyrhynchus cristatus) which were divided into five locus groups, Amcr-DAB1 through -DAB5, based on similarities along most of the coding and noncoding portions of the transcribed gene. All marine iguana sequences were monophyletic with respect to class II genes from other vertebrates indicating that they originated from a common ancestral locus after squamates split from other reptiles. The beta-1 domain, which is involved in antigen binding, exhibited signatures of positive selection as well as interlocus gene conversion in both long and short tracts-a pattern also observed in birds and fish, but not in mammals. On the other hand, the beta-2 domain was divergent between gene groups, which is characteristic of mammals. Based on these results, we preliminarily show that squamate class II B genes have been shaped by a unique blend of evolutionary forces that have been observed in differing degrees in other vertebrates.

Naive CD4(+) T cell populations that express TCRs specific for different foreign peptide-MHC class II complex (pMHCII) ligands can vary in size over several orders of magnitude. This variation may explain why immune responses to some peptides are stronger than others. In this study, we used a sensitive pMHCII-tetramer-based cell enrichment method to study the derivation of two naive foreign pMHCII-specific naive CD4(+) T cell populations that differed in size by 8-fold in normal mice. Analysis of mice in which thymic negative selection was impaired revealed that the smaller population underwent more clonal deletion than the larger population. In addition, large naive cell populations tended to recognize peptides with tryptophan residues as TCR contacts. Thus, the foreign pMHCII that tend to be recognized by large naive populations induce minimal clonal deletion and contain certain amino acids with the capacity to interact favorably with TCRs.

MHC gene products present antigenic epitopes to the antigen receptor on T cells. Nevertheless, direct binding of such epitopes to MHC class II proteins on normal living antigen-presenting cells (APCs) has not yet been demonstrated. We have previously shown a significant difference in the ability of T cells of myasthenia gravis (MG) patients to proliferate in response to the synthetic peptide p195-212 of the human acetylcholine receptor (AChR) alpha-subunit in comparison to healthy controls. The observed proliferative responses correlated significantly with HLA-DR5. Moreover, lymph node cells of various mouse strains that were primed with the T cell epitope, p195-212, were found to proliferate to different extents. To investigate these observations further, we designed an assay for direct binding of p195-212 to MHC class II proteins on the surface of freshly prepared splenic adherent cells. Binding of a biotinylated p195-212 was monitored using phycoerythrin-avidin by flow cytometry. Fifteen to sixty per cent of the cells were labeled following incubation with the biotinylated peptide. Binding was observed only to splenic adherent cells derived from mouse strains of which T cells were capable of proliferating in response to p195-212. The binding specificity, in terms of epitope structure and its site of interaction on the cells, was shown by its inhibition with an excess of the unlabeled peptide or with the relevant monoclonal anti-I-A antibodies. These results constitute the first direct evidence for the specific binding of a T cell epitope to live APC. PMID:2480232

Recombinant major histocompatibility complex (MHC) class II molecules were expressed with extracellular polypeptide domains reorganized to form heavy (H) and light (L) chains (alpha 1-beta 1-beta 2 and alpha 2) analogous to class I. Accurate protein folding and dimerization is demonstrated by the ability of this 3+1-DR1 construct to bind class II- restricted peptides and stimulate CD4+ T cells. Cell surface expression of a functional class II molecule consisting of H and L chains supports the validity of current class II models and affirms the evolutionary relatedness of class I/II. MHC functions that differ between class I/II may be influenced by domain configuration, and the use of domain- shifted constructs will allow examination of this possibility. PMID:8340763

Peptides bind MHC class II molecules through a thermodynamically nonadditive process consequent to the flexibility of the reactants. Currently, how the specific outcome of this binding process affects the ensuing epitope selection needs resolution. Calorimetric assessment of binding thermodynamics for hemagglutinin 306–319 peptide variants to the human MHC class II HLA-DR1 (DR1) and a mutant DR1 reveals that peptide/DR1 complexes can be formed with different enthalpic and entropic contributions. Complexes formed with a smaller entropic penalty feature circular dichroism spectra consistent with a non–compact form, and molecular dynamics simulation shows a more flexible structure. The opposite binding mode, compact and less flexible, is associated with greater entropic penalty. These structural variations are associated with rearrangements of residues known to be involved in HLA-DR (DM) binding, affinity of DM for the complex, and complex susceptibility to DM-mediated peptide exchange. Thus, the thermodynamic mechanism of peptide binding to DR1 correlates with the structural rigidity of the complex, and DM mediates peptide exchange by “sensing” flexible complexes in which the aforementioned residues are rearranged at a higher frequency than in more rigid ones. PMID:26116504

In North America, the raccoon rabies virus (RRV) is an endemic wildlife disease which causes acute encephalopathies and is a strong selective force on raccoons (Procyon lotor), with estimates of ∼85% of the population succumbing to the disease when epizootic. RRV is regarded as a lethal disease if untreated; therefore, no evolutionary response would be expected of raccoon populations. However, variable immune responses to RRV have been observed in raccoons indicating a potential for evolutionary adaptation. Studies of variation within the immunologically important major histocompatibility complex (MHC) have revealed relationships between MHC alleles and diseases in humans and other wildlife species. This enhances our understanding of how hosts and pathogens adapt and co-evolve. In this study, we used RRV as a model system to study host-pathogen interaction in raccoons from a challenge study and from four wild populations that differ in exposure times and viral lineages. We investigated the potential role of Prlo-DRB polymorphism in relation to susceptibility/resistance to RRV in 113 RRV positive and 143 RRV negative raccoons. Six alleles were found to be associated with RRV negative status and five alleles with RRV positive animals. We found variable patterns of MHC associations given the relative number of selective RRV sweeps in the studied regions and correlations between MHC diversity and RRV lineages. The allelic associations established provide insight into how the genetic variation of raccoons may affect the disease outcome and this can be used to examine similar associations between other rabies variants and their hosts.

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class IIMHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHCII and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHCII antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHCII (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHCII. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHCII. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHCII.

Glycosphingolipid- and cholesterol-rich membrane microdomains (rafts) in T-cells are important in triggering and regulation of T(H)-cell activation in immunological synapses (IS), which in turn may control the T-cell repertoire in lymph nodes and at the periphery. It is less known, however, how the "presynaptic side" controls formation and function of IS. We investigated here activation signals and synapse formation frequency of murine IP12-7 T(H) hybridoma cell specific to influenza virus HA-peptide upon stimulation with two B-lymphoma cells, A20 and 2PK3, pulsed with peptide antigen. Confocal microscopic colocalization and FRET data consonantly revealed clustered distribution and constitutive raft-association of a major fraction of MHC-II molecules in both APCs. Costimulatory molecules (CD80 and CD86), not associated constitutively with rafts, were expressed at much lower level in A20 cells. T-cells responded to 2PK3 APC with much higher signal strength than to A20 cells, in good correlation with the frequency of IS formation, as assessed by microscopic conjugation assay. Disruption of rafts by cholesterol depletion in 2PK3 cells largely decreased the magnitude of T(H) cell activation signals, especially at low peptide antigen doses, similarly to masking CD4 with mAb on T-cells. The frequency of IS formation was reduced by blocking LFA-1 on T-cells and CD80 on APCs, by lowering the temperature below the phase transition of the membrane or by disrupting actin cytoskeleton. These data together suggest that the surface density and affinity/stability of peptide-MHC-II complexes and the costimulatory level are primary determinants for an efficient TCR recognition and the strength of the subsequent T-cell signals, as well as of the IS formation, which additionally requires a cytoskeleton-dependent remodeling of APC surface after the initial TCR signal. The threshold of T-cell activation can be further set by rafting MHC-II domains via concentrating high affinity

Background The histocompatibility complex (MHC) class I expression in the central nervous system (CNS) regulates synaptic plasticity events during development and adult life. Its upregulation may be associated with events such as axotomy, cytokine exposition and changes in neuron electrical activity. Since IFNγ is a potent inducer of the MHC I expression, the present work investigated the importance of this pro-inflammatory cytokine in the synaptic elimination process in the spinal cord, as well as the motor recovery of IFN−/−, following peripheral injury. Methods The lumbar spinal cords of C57BL/6J (wild type) and IFNγ−/− (mutant) mice, subjected to unilateral sciatic nerve transection, were removed and processed for immunohistochemistry and real time RT-PCR, while the sciatic nerves from animals subjected to unilateral crush, were submitted to immunohistochemistry and electron microscopy for counting of the axons. Gait recovery was monitored using the Cat Walk system. Newborn mice astrocyte primary cultures were established in order to study the astrocytic respose in the absence of the IFNγ expression. Results IFNγ−/− mutant mice showed a decreased expression of MHC I and β2-microglobulin mRNA coupled with reduced synaptophysin immunolabelling in the lesioned spinal cord segment. Following unilateral nerve transection, the Iba-1 (ionized calcium binding adaptor molecule 1) and glial fibrillary acid protein (GFAP) reactivities increased equally in both strains. In vitro, the astrocytes demonstrated similar GFAP levels, but the proliferation rate was higher in the wild type mice. In the crushed nerves (distal stump), neurofilaments and p75NTR immunolabeling were upregulated in the mutant mice as compared to the wild type and an improvement in locomotor recovery was observed. Conclusion The present results show that a lack of IFNγ affects the MHC I expression and the synaptic elimination process in the spinal cord. Such changes, however, do not

The Lewis rat, an inbred rat strain susceptible to several well-characterized experimental autoimmune diseases, provides a good model to study peptide-mediated immunotherapy. Peptide immunotherapy focussing on the modulation of T cell responses by interfering with TCR-peptide-MHC complex formation requires the elucidation of the molecular basis of TCR-peptide-MHC interactions for an efficient design of modulatory peptides. In the Lewis rat most autoimmune-associated CD4+ T cell responses are MHC class II RT1.BL restricted. In this study, the characteristics of RT1.BL-peptide interactions were explored. A series of substitution analogs of two Lewis rat T cell epitopes was examined in a direct peptide-MHC binding assay on isolated RT1.BL molecules. Furthermore, other autoimmune-related as well as non-disease-related T cell epitopes were tested in the binding assay. This has led to the definition of an extended RT1.BL-peptide binding motif. The RT1.BL-peptide binding motif established in this study is the first described rat MHC-peptide binding motif based on direct MHC-peptide binding experiments. To predict good or intermediate RT1.BL binding peptides, T cell epitope search profiles were deduced from this motif. The motif and search profiles will greatly facilitate the prediction of modulatory peptides based on autoimmune-associated T cell epitopes and the identification of target structures in experimental autoimmune diseases in Lewis rats.

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D(b) mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D(b) in P15 mice. At E10.5, the earliest stage we examined, H2-D(b) was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D(b) expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D(b) mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D(b) in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K(b) signal paralleled that of H2-D(b) and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D(b) at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D(b) in the developing CNS.

Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms a large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.

Here we present the sequence of a 45 kb cosmid containing a previously characterized poly-morphic Mhc class II B gene (Agph-DAB1) from the red-winged blackbird (Agelaius phoeniceus). We compared it with a previously sequenced cosmid from this species, revealing two regions of 7.5 kb and 13.0 kb that averaged greater than 97% similarity to each another, indicating a very recent shared duplication. We found 12 retroelements, including two chicken repeat 1 (CR1) elements, constituting 6.4% of the sequence and indicating a lower frequency of retroelements than that found in mammalian genomic DNA. Agph-DAB3, a new class II B gene discovered in the cosmid, showed a low rate of polymorphism and may be functional. In addition, we found a Mhc class II B gene fragment and three genes likely to be functional (encoding activin receptor type II, a zinc finger, and a putative gamma-filamin). Phylogenetic analysis of exon 2 alleles of all three known blackbird Mhc genes indicated strong clustering of alleles by locus, implying that large amounts of interlocus gene conversion have not occurred since these genes have been diverging. Despite this, interspecific comparisons indicate that all three blackbird Mhc genes diverged from one another less than 35 million years ago and are subject to concerted evolution in the long term. Comparison of blackbird and chicken Mhc promoter regions revealed songbird promoter elements for the first time. The high gene density of this cosmid confirms similar findings for the chicken Mhc, but the segment duplications and diversity of retroelements resembles mammalian sequences.

In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.

Suppression of CD8+ T cell activation is a critical mechanism used by Mycobacterium tuberculosis (MTB) to escape protective host immune responses. PPE38 belongs to the unique PPE family of MTB and in our previous study, PPE38 protein was speculated to participate in manipulating macrophage MHC class I pathway. To test this hypothesis, the function of mycobacterial PPE38 protein was assessed here using macrophage and mouse infection models. Decreased amount of MHC class I was observed on the surface of macrophages infected with PPE38-expressing mycobacteria. The transcript of genes encoding MHC class I was also inhibited by PPE38. After infection of C57BL/6 mice with Mycobacterium smegmatis expressing PPE38 (Msmeg-PPE38), decreased number of CD8+ T cells was found in spleen, liver, and lungs through immunohistochemical analysis, comparing to the control strain harboring empty vector (Msmeg-V). Consistently, flow cytometry assay showed that fewer effector/memory CD8+ T cells (CD44highCD62Llow) were activated in spleen from Msmeg-PPE38 infected mice. Moreover, Msmeg-PPE38 confers a growth advantage over Msmeg-V in C57BL/6 mice, indicating an effect of PPE38 to favor mycobacterial persistence in vivo. Overall, this study shows a unique biological function of PPE38 protein to facilitate mycobacteria to escape host immunity, and provides hints for TB vaccine development. PMID:28348981

Evidence has recently emerged that butyrophilins, which are members of the extended B7 family of co-stimulatory molecules, have diverse functions in the immune system. We found that the human and mouse genes encoding butyrophilin-2A2 (BTN2A2) are regulated by the class II trans-activator and regulatory factor X, two transcription factors dedicated to major histocompatibility complex class IIexpression, suggesting a role in T cell immunity. To address this, we generated Btn2a2-deficient mice. Btn2a2−/− mice exhibited enhanced effector CD4+ and CD8+ T cell responses, impaired CD4+ regulatory T cell induction, potentiated antitumor responses, and exacerbated experimental autoimmune encephalomyelitis. Altered immune responses were attributed to Btn2a2 deficiency in antigen-presenting cells rather than T cells or nonhematopoietic cells. These results provide the first genetic evidence that BTN2A2 is a co-inhibitory molecule that modulates T cell–mediated immunity. PMID:26809444

Monocytes and DCs originate from a shared precursor in the bone marrow, and steady-state DCs in lymphoid organs develop directly from the precursor rather than via a monocyte intermediate. However, monocytes can differentiate into DCs in tissues such as the lung and gut mucosa and into macrophages in most tissues. As Ly6C hi monocytes accumulate in lymphoid organs during oral Salmonella infection, we investigated their ability to develop into potential DCs, identified as CD11c hi MHC-II hi cells, in infected hosts. Ly6C hi monocytes, isolated from the blood of Salmonella-infected mice, developed into CD11c hi MHC-II hi cells after culture with GM-CSF or Flt3L. In contrast, the same monocytes cultured in the presence of GM-CSF and heat-killed Salmonella did not differentiate into CD11c hi MHC-II hi cells. The bacteria-induced differentiation block was dependent on TLRs, as monocytes from MyD88-/- mice converted into CD11c hi MHC-II hi cells even in the presence of bacteria. We hypothesized that Salmonella-activated wild-type monocytes secreted mediators that inhibited differentiation of MyD88-/--derived monocytes. However, IL-6, IL-10, TNF-alpha, or IL-12p70 did not account for the inhibition. Finally, monocyte-derived CD11c hi MHC-II hi cells pulsed with OVA peptide or protein did not induce proliferation of antigen-specific CD4+ T cells but rather, suppressed the ability of DCs to activate CD4+ T cells. Overall, the data show that Ly6C hi monocytes from Salmonella-infected mice develop into CD11c hi MHC-II hi cells with poor antigen-presentation capacity when cultured ex vivo, and that monocyte exposure to Salmonella inhibits their differentiation into CD11c hi MHC-II hi cells in a MyD88-dependent fashion.

The Major Histocompatibility Complex (Mhc) class II DRB locus of vertebrates is highly polymorphic and some alleles may be shared between closely related species as a result of balancing selection in association with resistance to parasites. In this study, we developed a new set of PCR primers to amplify, clone, and sequence overlapping portions of the Mhc class II DRB-like gene from the 5'UTR end to intron 3, including exons 1, 2, and 3 and introns 1 and 2 in four species (20 Humboldt, six African, five Magellanic, and three Galapagos penguins) of penguin from the genus Spheniscus (Sphe). Analysis of gene sequence variation by the neighbor-joining method of 21 Sphe sequences and 20 previously published sequences from four other penguin species revealed overlapping clades within the Sphe species, but species-specific clades for the other penguin species. The overlap of the DRB-like gene sequence variants between the four Sphe species suggests that, despite their allopatric distribution, the Sphe species are closely related and that some shared DRB1 alleles may have undergone a trans-species inheritance because of balancing selection and/or recent rapid speciation. The new primers and PCR assays that we have developed for the identification of the DRB1 DNA and protein sequence variations appear to be useful for the characterization of the molecular evolution of the gene in closely related Penguin species and might be helpful for the assessment of the genetic health and the management of the conservation and captivity of these endangered species.

The 15 extant species of Darwin's finches on the Galápagos and Cocos Islands are the products of an unfinished adaptive radiation from a founder flock of birds related to the South American species Tiaris obscura. Molecular characterization of their major histocompatibility complex ( Mhc) class II B genes has revealed the existence of several related groups of sequences (presumably encoded in distinct loci) from which one (group 5) stands out because of its low divergence over extended time periods. Analysis of group 5 exon 2 and intron 2 sequences has revealed that the encoding locus apparently arose 2-3 million years ago in the Tiaris group of South and Central American Thraupini. The locus shows no evidence of inactivation, but displays a very low degree of polymorphism, both in terms of number of alleles and genetic distances between alleles. Some of the polymorphism, however, appears to be trans-specific. All the observed intergenic differences can be explained by point mutations and most of the exon 2 changes represent non-synonymous substitutions, although the rate of non-synonymous and synonymous substitutions appears to be the same. The origin of the new locus is explained by the birth-and-death model of Mhc evolution with two important extensions. First, the ancestor of the group 5 genes may have arisen without new gene duplication and second, the birth of the new group may have been brought about by a switch from balancing to directional selection. The ancestor of the group 5 genes may have been a classical class II B allele (one of many) which directional selection fixed in the ancestral population and drove into the category of nonclassical genes.

Major histocompatibility complex class II (MHCII) and canine distemper virus (CDV) antigen expression were compared by immunohistochemistry in the cerebellar white matter of ten dogs with naturally occurring canine distemper encephalitis. In addition, infiltrating mononuclear cells were characterized by employing poly- and monoclonal antibodies directed against human CD3, canine MHCII, CD5, B cell antigen and CDV-specific nucleoprotein. Positive antigen-antibody reaction was visualized by the avidin-biotin-peroxidase complex method on frozen sections. Histologically, neuropathological changes were categorized into acute, subacute, and chronic. In control brains, MHCIIexpression was weak and predominantly detected on resident microglia of the white matter and on endothelial, perivascular and intravascular cells. In CDV antigen-positive brains, MHCII was mainly found on microglia and to a lesser extent on endothelial, meningeal, choroid plexus epithelial, ependymal and intravascular cells. In addition, virtually all of the perivascular cells expressedMHCII antigen. CDV antigen was demonstrated most frequently in astrocytes. Of the perivascular lymphocytes, the majority were CD3-positive cells, followed by B cells. Only a small proportion of perivascular cells expressed the CD5 antigen. In addition, B cells and CD3 and CD5 antigen-positive cells were found occasionally in subacute and frequently in chronic demyelinating plaques. In acute encephalitis, CDV antigen exhibited a multifocal or diffuse distribution, and MHCII was moderately up-regulated throughout the white matter and accentuated in CDV antigen-positive plaques. In subacute encephalitis, moderate multifocal CDV antigen and moderate to strong diffuse MHCII-specific staining, especially prominent in CDV antigen-positive lesions, were observed. In chronic encephalitis, CDV antigen expression was restricted to single astrocytes at the edge of the lesions or was absent, while MHCIIexpression

Tibetan macaque (Macaca thibetana), an endangered primate species endemic to China, have been used as experimental animal model for various human diseases. Major histocompatibility complex (MHC) genes play a crucial role in the susceptibility and/or resistance to many human diseases, but little is known about Tibetan macaques. To gain an insight into the MHC background and to facilitate the experimental use of Tibetan macaques, the second exon of Mhc-DQB1 gene was sequenced in a cohort of wild Tibetan macaques living in the Sichuan province of China. A total of 23 MhcMath-DQB1 alleles were identified for the first time, illustrating a marked allelic polymorphism at the DQB1 locus for these macaques. Most of the sequences (74%) observed in this study belong to DQB1*06 (9 alleles) and DQB1*18 (8 alleles) lineages, and the rest (26%) belong to DQB1*15 (3 alleles) and DQB1*17 (3 alleles) lineages. The most frequent alleles detected among these macaques were MhcMath-DQB1*15:02:02 (17.9%), followed by Math-DQB1*06:06, 17:03 and 18:01, which were detected in 9 (16.1%) of the monkeys, respectively. Non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions in the peptide-binding region, suggesting balancing selection for maintaining polymorphisms at the MHC class II DQB1 locus. Phylogenetic analyses confirms the trans-species model of evolution of the Mhc-DQB1 genes in non-human primates, and in particular, the extensive allele sharing is observed between Tibetan and other macaque species.

The presentation of peptide-MHCII complexes (pMHCIIs) for surveillance by T cells is a well-known immunological concept in vertebrates, yet the conformational dynamics of antigen exchange remain elusive. By combining NMR-detected H/D exchange with Markov modelling analysis of an aggregate of 275 microseconds molecular dynamics simulations, we reveal that a stable pMHCII spontaneously samples intermediate conformations relevant for peptide exchange. More specifically, we observe two major peptide exchange pathways: the kinetic stability of a pMHCII's ground state defines its propensity for intrinsic peptide exchange, while the population of a rare, intermediate conformation correlates with the propensity of the HLA-DM-catalysed pathway. Helix-destabilizing mutants designed based on our model shift the exchange behaviour towards the HLA-DM-catalysed pathway and further allow us to conceptualize how allelic variation can shape an individual's MHC restricted immune response. PMID:27827392

Major histocompatibility complex (MHC) molecules are key players in initiating immune responses towards invading pathogens. Both MHC class I and class II genes are present in teleosts, and, using phylogenetic clustering, sequences from both classes have been classified into various lineages. The polymorphic and classical MHC class I and class II gene sequences belong to the U and A lineages, respectively. The remaining class I and class II lineages contain nonclassical gene sequences that, despite their non-orthologous nature, may still hold functions similar to their mammalian nonclassical counterparts. However, the fact that several of these nonclassical lineages are only present in some teleost species is puzzling and questions their functional importance. The number of genes within each lineage greatly varies between teleost species. At least some gene expansions seem reasonable, such as the huge MHC class I expansion in Atlantic cod that most likely compensates for the lack of MHC class II and CD4. The evolutionary trigger for similar MHC class I expansions in tilapia, for example, which has a functional MHC class II, is not so apparent. Future studies will provide us with a more detailed understanding in particular of nonclassical MHC gene functions. PMID:26797646

The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-gamma)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-IIexpression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-IIexpression, indicating that any Brucella lipoprotein could down-modulate MHC-IIexpression and Ag processing. Inhibition of MHC-IIexpression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-IIexpression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-gamma production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection.

For more than two decades, the cheetah (Acinonyx jubatus) has been considered a paradigm of disease vulnerability associated with low genetic diversity, particularly at the immune genes of the major histocompatibility complex (MHC). Cheetahs have been used as a classic example in numerous conservation genetics textbooks as well as in many related scientific publications. However, earlier studies used methods with low resolution to quantify MHC diversity and/or small sample sizes. Furthermore, high disease susceptibility was reported only for captive cheetahs, whereas free-ranging cheetahs show no signs of infectious diseases and a good general health status. We examined whether the diversity at MHC class I and class II-DRB loci in 149 Namibian cheetahs was higher than previously reported using single-strand conformation polymorphism analysis, cloning, and sequencing. MHC genes were examined at the genomic and transcriptomic levels. We detected ten MHC class I and four class II-DRB alleles, of which nine MHC class I and all class II-DRB alleles were expressed. Phylogenetic analyses and individual genotypes suggested that the alleles belong to four MHC class I and three class II-DRB putative loci. Evidence of positive selection was detected in both MHC loci. Our study indicated that the low number of MHC class I alleles previously observed in cheetahs was due to a smaller sample size examined. On the other hand, the low number of MHC class II-DRB alleles previously observed in cheetahs was further confirmed. Compared with other mammalian species including felids, cheetahs showed low levels of MHC diversity, but this does not seem to influence the immunocompetence of free-ranging cheetahs in Namibia and contradicts the previous conclusion that the cheetah is a paradigm species of disease vulnerability.

Generation of tumor-antigen specific CD4(+) T-helper (T(H)) lines through in vitro priming is of interest for adoptive cell therapy of cancer, but the development of this approach has been limited by the lack of appropriate tools to identify and isolate low frequency tumor antigen-specific CD4(+) T cells. Here, we have used recently developed MHC class II/peptide tetramers incorporating an immunodominant peptide from NY-ESO-1 (ESO), a tumor antigen frequently expressed in different human solid and hematologic cancers, to implement an in vitro priming platform allowing the generation of ESO-specific T(H) lines. We isolated phenotypically defined CD4(+) T-cell subpopulations from circulating lymphocytes of DR52b(+) healthy donors by flow cytometry cell sorting and stimulated them in vitro with peptide ESO(119-143), autologous APC and IL-2. We assessed the frequency of ESO-specific cells in the cultures by staining with DR52b/ESO(119-143) tetramers (ESO-tetramers) and TCR repertoire of ESO-tetramer(+) cells by co-staining with TCR variable β chain (BV) specific antibodies. We isolated ESO-tetramer(+) cells by flow cytometry cell sorting and expanded them with PHA, APC and IL-2 to generate ESO-specific T(H) lines. We characterized the lines for antigen recognition, by stimulation with ESO peptide or recombinant protein, cytokine production, by intracellular staining using specific antibodies, and alloreactivity, by stimulation with allo-APC. Using this approach, we could consistently generate ESO-tetramer(+) T(H) lines from conventional CD4(+)CD25(-) naïve and central memory populations, but not from effector memory populations or CD4(+)CD25(+) Treg. In vitro primed T(H) lines recognized ESO with affinities comparable to ESO-tetramer(+) cells from patients immunized with an ESO vaccine and used a similar TCR repertoire. In this study, using MHC class II/ESO tetramers, we have implemented an in vitro priming platform allowing the generation of ESO

Upon antigen (Ag) encounter, B cells require T-cell help to enter the germinal center (GC). They obtain this help by presenting Ag-derived peptides on MHC class II (MHCII) for recognition by the T-cell receptor (TCR) of CD4(+) T cells. Peptides are loaded onto MHCII in endosomal compartments in a process catalyzed by the MHCII-like protein H2-M (HLA-DM in humans). This process is modulated by another MHCII-like protein, H2-O (HLA-DO in humans). H2-O is a biochemical inhibitor of peptide loading onto MHCII; however, on the cellular level, it has been shown to have varying effects on Ag presentation. Thus, the function of H2-O in the adaptive immune response remains unclear. Here, we examine the effect of H2-O expression on the ability of Ag-specific B cells to enter the GC. We show that when Ag specific WT and H2-O(-/-) B cells are placed in direct competition, H2-O(-/-) B cells preferentially populate the GC. This advantage is confined to Ag-specific B cells and is due to their superior ability to obtain Ag-specific T-cell help when T-cell help is limiting. Overall, our work shows that H2-O expression reduces the ability of B cells to gain T-cell help and participate in the GC reaction.

To further our understanding of the evolution of avian Mhc genes at the genomic level, we screened a cosmid library made from a red-winged blackbird (Agelaius phoeniceus) with a blackbird cDNA probe and subcloned from one of the Mhc-containing cosmids a gene which we designate Agph-DAB1. The structure of the gene is similar to that found for chicken class II B genes, except that the introns are surprisingly large, ranging from 98 to over 600 bp, making this the longest avian class II B gene to date. Using primers targeted toward the introns flanking the peptide-binding region (PBR), we amplified the entirety of the second exon and determined nucleotide sequences of 41 PCR products from eight individual blackbirds. The 10 sequence types found, among which were two probable pseudogene sequences, exhibit the classic hallmarks for evolution of PBRs, namely, an excess of nonsynonymous over synonymous substitutions and evidence of gene conversion events in polymorphic subdomains. Despite these patterns and our use of intron primers, the distribution of sequences among individuals suggests that more than one locus was amplified in most individuals, and the bushlike tree of sequences provides little information as to locus-specific clusters. These results imply a complex history of gene conversion, recent duplication, or possibly, concerted evolution among multiple loci, although Agph-DAB1, the first genomic Mhc sequence from a bird other than chicken, provides important clues in the quest for locus-specific Mhc primers in birds.

Here, we sought to determine whether peptide vaccines designed harbor both class I as well as class II restricted antigenic motifs could concurrently induce CD4 and CD8 T cell activation against autologous tumor antigens. Based on our prior genome-wide interrogation of human prostate cancer tissues to identify genes over-expressed in cancer and absent in the periphery, we targeted SIM2 as a prototype autologous tumor antigen for these studies. Using humanized transgenic mice we found that the 9aa HLA-A*0201 epitope, SIM2(237-245), was effective at inducing an antigen specific response against SIM2-expressing prostate cancer cell line, PC3. Immunization with a multi-epitope peptide harboring both MHC-I and MHC-II restricted epitopes induced an IFN-γ response in CD8 T cells to the HLA-A*0201-restricted SIM2(237-245) epitope, and an IL-2 response by CD4 T cells to the SIM2(240-254) epitope. This peptide was also effective at inducing CD8+ T-cells that responded specifically to SIM2-expressing tumor cells. Collectively, the data presented in this study suggest that a single peptide containing multiple SIM2 epitopes can be used to induce both a CD4 and CD8 T cell response, providing a peptide-based vaccine formulation for potential use in immunotherapy of various cancers.

Hypocrellin A has gained much attention in recent years due to its light-induced antitumor, antifungal and antiviral activities. Here we report that hypocrellin A exerts immunomodulatory effects on MHC-restricted presentation of antigen. Hypocrellin A inhibited class II-MHC restricted presentation of exogenous antigen, but not class I MHC-restricted presentation of exogenous antigen, in dendritic cells. Hypocrellin A also inhibited the cytosolic pathway of endogenous antigen presentation. However, hypocrellin A did not inhibit the expression of class I and class IIMHC molecules on dendritic cells (DCs), the phagocytic activity of DCs, or the H-2Kb-restricted presentation of a synthetic peptide, SIINFEKL. These results show that hypocrellin A differentially modulates the MHC-restricted antigen presentation pathways. PMID:22346783

We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales (Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures.

The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales (Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures. PMID:26761201

Major histocompatibility complex (MHC) genes encode proteins that play a critical role in vertebrate immune system and are highly polymorphic. To further understand the molecular evolution of the MHC genes, we compared MHC class II DRB genes between the Japanese weasel (Mustela itatsi), a species endemic to Japan, and the Siberian weasel (Mustela sibirica), a closely related species on the continent. We sequenced a 242-bp region of DRB exon 2, which encodes antigen-binding sites (ABS), and found 24 alleles from 31 M. itatsi individuals and 17 alleles from 21 M. sibirica individuals, including broadly distributed, species-specific and/or geographically restricted alleles. Our results suggest that pathogen-driven balancing selection have acted to maintain the diversity in the DRB genes. For predicted ABS, nonsynonymous substitutions exceeded synonymous substitutions, also indicating positive selection, which was not seen at non-ABS. In a Bayesian phylogenetic tree, two M. sibirica DRB alleles were basal to the rest of the sequences from mustelid species and may represent ancestral alleles. Trans-species polymorphism was evident between many mustelid DRB alleles, especially between M. itatsi and M. sibirica. These two Mustela species divided about 1.7 million years ago, but still share many MHC alleles, indicative of their close phylogenetic relationship.

The blood-brain barrier (BBB) normally bars peripheral T lymphocytes from entering the cerebrum. Interestingly, activated T cells exist as infiltrates in the brains of Alzheimer's disease (AD) patients, but little is known about the mechanisms involved. In this study, we observed significantly higher MHC class I expression in rat brain endothelial cells compared with controls following the induction of experimental AD models. An in vitro BBB model, which was constructed with human brain microvascular endothelial cells, was established to study the mechanisms underlying the transendothelial migration of T cells. Using in vitro studies, we demonstrated that secretion of TNF-α from Aβ1-42-treated BV2 microglia contributes to the elevated expression of MHC class I on the brain microvessel endothelium. Transmigration assays and adhesion assays confirmed that the upregulation of MHC class I molecules was associated with T cell transendothelial migration. MHC class I knock-down in HBMECs significantly attenuated the migratory and adhesive capability of the T cells. Interestingly, a TNF-α neutralizing antibody effectively blocked the transendothelial migration of T cells triggered by treatment with the supernatant from Aβ1-42-treated BV2 microglia. We propose that microglia-derived TNF-α upregulates MHC class I molecule expression on brain endothelial cells, which represents a mechanism of T cell migration into the brain. This study may provide a new insight into the potential pathomechanism of Alzheimer's disease.

Heterogeneities in immune responsiveness may affect key epidemiological parameters and the dynamics of pathogens. The roles of immunogenetics in these variations remain poorly explored. We analysed the influence of Major histocompatibility complex (Mhc) genes and epigamic traits on the response to phytohaemagglutinin in males from cyclic populations of the montane water vole (Arvicola scherman). Besides, we tested the relevance of lateral scent glands as honest signals of male quality. Our results did not corroborate neither the hypotheses of genome-wide heterozygosity-fitness correlation nor the Mhc heterozygote advantage. We found a negative relationship between Mhc hetetozygosity and response to phytohaemagglutinin, mediated by a specific Mhc homozygous genotype. Our results therefore support the hypothesis of the Arte-Dqa-05 homozygous genotype being a ‘good’ Mhc variant in terms of immunogenetic quality. The development of the scent glands seems to be an honest signal for mate choice as it is negatively correlated with helminth load. The ‘good gene’ hypothesis was not validated as Arte-Dqa-05 homozygous males did not exhibit larger glands. Besides, the negative relationship observed between the size of these glands and the response to phytohaemagglutinin, mainly for Mhc homozygotes, corroborates the immunocompetence handicap hypothesis. The Mhc variants associated with larger glands remain yet to be determined. PMID:25567924

Heterogeneities in immune responsiveness may affect key epidemiological parameters and the dynamics of pathogens. The roles of immunogenetics in these variations remain poorly explored. We analysed the influence of Major histocompatibility complex (Mhc) genes and epigamic traits on the response to phytohaemagglutinin in males from cyclic populations of the montane water vole (Arvicola scherman). Besides, we tested the relevance of lateral scent glands as honest signals of male quality. Our results did not corroborate neither the hypotheses of genome-wide heterozygosity-fitness correlation nor the Mhc heterozygote advantage. We found a negative relationship between Mhc hetetozygosity and response to phytohaemagglutinin, mediated by a specific Mhc homozygous genotype. Our results therefore support the hypothesis of the Arte-Dqa-05 homozygous genotype being a 'good' Mhc variant in terms of immunogenetic quality. The development of the scent glands seems to be an honest signal for mate choice as it is negatively correlated with helminth load. The 'good gene' hypothesis was not validated as Arte-Dqa-05 homozygous males did not exhibit larger glands. Besides, the negative relationship observed between the size of these glands and the response to phytohaemagglutinin, mainly for Mhc homozygotes, corroborates the immunocompetence handicap hypothesis. The Mhc variants associated with larger glands remain yet to be determined.

Major histocompatibility class II (MHCII) molecules are loaded with peptides derived from foreign and self-proteins within the endosomes and lysosomes of antigen presenting cells (APCs). This process is mediated by interaction of MHCII with the conserved, non-polymorphic MHCII like molecule HLA-DM (DM). DM activity is directly opposed by HLA-DO (DO), another conserved, non-polymorphic MHCII like molecule. DO is an MHCII substrate mimic. Binding of DO to DM prevents MHCII from binding to DM, thereby inhibiting peptide loading. Inhibition of DM function enables low stability MHC complexes to survive and populate the surface of APCs. As a consequence, DO promotes the display of a broader pool of low abundance self-peptides. Broadening the peptide repertoire theoretically reduces the likelihood of inadvertently acquiring a density of self-ligands that is sufficient to activate self-reactive T cells. One function of DO, therefore, is to promote T cell tolerance by shaping the visible image of self. Recent data also shows that DO influences the adaptive immune response by controlling B cell entry into the germinal center reaction. This review explores the data supporting these concepts.

Major histocompatibility class II (MHCII) molecules are loaded with peptides derived from foreign and self-proteins within the endosomes and lysosomes of antigen presenting cells (APCs). This process is mediated by interaction of MHCII with the conserved, non-polymorphic MHCII like molecule HLA-DM (DM). DM activity is directly opposed by HLA-DO (DO), another conserved, non-polymorphic MHCII like molecule. DO is an MHCII substrate mimic. Binding of DO to DM prevents MHCII from binding to DM, thereby inhibiting peptide loading. Inhibition of DM function enables low stability MHC complexes to survive and populate the surface of APCs. As a consequence, DO promotes the display of a broader pool of low abundance self-peptides. Broadening the peptide repertoire theoretically reduces the likelihood of inadvertently acquiring a density of self-ligands that is sufficient to activate self-reactive T cells. One function of DO, therefore, is to promote T cell tolerance by shaping the visible image of self. Recent data also shows that DO influences the adaptive immune response by controlling B cell entry into the germinal center reaction. This review explores the data supporting these concepts. PMID:24381574

Whooping cough is currently seeing resurgence in countries despite high vaccine coverage. There is considerable variation in subject-specific response to infection and vaccine efficacy, but little is known about the role of human genetics. We carried out a case-control genome-wide association study of adult or parent-reported history of whooping cough in two cohorts from the UK: the ALSPAC cohort and the 1958 British Birth Cohort (815/758 cases and 6341/4308 controls, respectively). We also imputed HLA alleles using dense SNP data in the MHC region and carried out gene-based and gene-set tests of association and estimated the amount of additive genetic variation explained by common SNPs. We observed a novel association at SNPs in the MHC class II region in both cohorts [lead SNP rs9271768 after meta-analysis, odds ratio [95% confidence intervals (CIs)] 1.47 (1.35, 1.6), P-value 1.21E - 18]. Multiple strong associations were also observed at alleles at the HLA class II loci. The majority of these associations were explained by the lead SNP rs9271768. Gene-based and gene-set tests and estimates of explainable common genetic variation could not establish the presence of additional associations in our sample. Genetic variation at the MHC class II region plays a role in susceptibility to whooping cough. These findings provide additional perspective on mechanisms of whooping cough infection and vaccine efficacy.

Anti-CD44 or anti-MHCII antibodies bound to tissue culture plates have previously been shown to induce a dramatic generation of dendritic processes in activated murine B cells. In this study, we demonstrate a similar generation of dendrites and cell motility in activated B cells through CD45R. The dynamic formation of dendritic processes and associated induction of cell motility were analyzed by video microscopy and were characterized by a rapid, and multidirectional emission of dendrites with retractile behavior. The addition of cytochalasin E totally blocked dendrites formation and motility induced through either CD45R, CD44 or MHCII, suggesting that the necessary cytoskeletal rearrangements require active polymerization of actin. Confocal microscopy showed an accumulation of F-actin in the dendrites, as long as cells were elongating. In contrast, G-actin was localized in the perinuclear area and also accumulated in sites where dendrites originated. Preincubation of B cells with staurosporine (a PKC inhibitor) or BAPTA-AM (a calcium chelator) prevented these morphological changes, indicating additionally a requirement for a PKC-calcium-dependent activity. Dendrite formation and cellular motility, therefore, seem to be two manifestations of the same phenomenon, and CD44, CD45R and MHCII appear to be signaling molecules for the observed cytoskeleton-dependent morphological changes.

ABSTRACT Major histocompatibility complex class II (MHC-II) molecules play a central role in adaptive antiviral immunity by presenting viral peptides to CD4+ T cells. Due to their key role in adaptive immunity, many viruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), have evolved multiple strategies to inhibit the MHC-II antigen presentation pathway. The expression of MHC-II, which is controlled mainly at the level of transcription, is strictly dependent upon the binding of the class II transactivator (CIITA) to the highly conserved promoters of all MHC-II genes. The recruitment of CIITA to MHC-II promoters requires its direct interactions with a preassembled MHC-II enhanceosome consisting of cyclic AMP response element-binding protein (CREB) and nuclear factor Y (NF-Y) complex and regulatory factor X (RFX) complex proteins. Here, we show that KSHV-encoded latency-associated nuclear antigen (LANA) disrupts the association of CIITA with the MHC-II enhanceosome by binding to the components of the RFX complex. Our data show that LANA is capable of binding to all three components of the RFX complex, RFX-associated protein (RFXAP), RFX5, and RFX-associated ankyrin-containing protein (RFXANK), in vivo but binds more strongly with the RFXAP component in in vitro binding assays. Levels of MHC-II proteins were significantly reduced in KSHV-infected as well as LANA-expressing B cells. Additionally, the expression of LANA in a luciferase promoter reporter assay showed reduced HLA-DRA promoter activity in a dose-dependent manner. Chromatin immunoprecipitation assays showed that LANA binds to the MHC-II promoter along with RFX proteins and that the overexpression of LANA disrupts the association of CIITA with the MHC-II promoter. These assays led to the conclusion that the interaction of LANA with RFX proteins interferes with the recruitment of CIITA to MHC-II promoters, resulting in an inhibition of MHC-II gene expression. Thus, the data presented here identify

The products of the class II-like H2-M genes of the major histocompatibility complex are required for class II antigen processing. We sequenced H2-Ma and Mb from several mouse strains to determine whether these genes are polymorphic like the classical H2-A and E genes, or are oligomorphic, like H2-O. Both Mb loci appear to be transcribed and are distinct from each other. Mb1 and Mb2 differ by about 11% at the nucleotide level and are most dissimilar in their second exons (corresponding to the beta 1 domain). Relative to the published Mb1d haplotype sequence, the products of the b, g7, f, and k2 alleles of Mb1 from Mus musculus domesticus and the separate mouse species Mus spretus differ by only one to four amino acids. The majority of the changes occurred in the second exon of Mb1, in contrast to HLA-DMB, the human orthologue. Little polymorphism was seen for Mb2, and Ma was invariant in all strains tested. The similarity of the g7 allele to those from other haplotypes makes it unlikely that the M class II genes play a role in the autoimmune diabetes of NOD strain mice. The M genes are regulated in a manner similar to classical class II genes, in that they are upregulated by IFN-gamma in macrophages, and to a lesser extent by IL4 in B cells. When modeled on the crystal structure of the HLA-DR1 class II molecule, nearly all of the differences between M beta 1 and M beta 2 affect residues facing away from the putative peptide binding groove.

Immune-evasion and immune checkpoints are promising new therapeutic targets for several cancer entities. In ovarian cancer, the clinical role of programmed cell death receptor ligand 1 (PD-L1) expression as mechanism to escape immune recognition has not been clarified yet. We analyzed PD-L1 expression of primary ovarian and peritoneal tumor tissues together with several other parameters (whole transcriptomes of isolated tumor cells, local and systemic immune cells, systemic cytokines and metabolites) and compared PD-L1 expression between primary tumor and tumor recurrences. All expressed major histocompatibility complex (MHC) I genes were negatively correlated to PD-L1 abundances on tumor tissues, indicating two mutually exclusive immune-evasion mechanisms in ovarian cancer: either down-regulation of T-cell mediated immunity by PD-L1 expression or silencing of self-antigen presentation by down-regulation of the MHC I complex. In our cohort and in most of published evidences in ovarian cancer, low PD-L1 expression is associated with unfavorable outcome. Differences in immune cell populations, cytokines, and metabolites strengthen this picture and suggest the existence of concurrent pathways for progression of this disease. Furthermore, recurrences showed significantly increased PD-L1 expression compared to the primary tumors, supporting trials of checkpoint inhibition in the recurrent setting. PMID:28266500

Class II major histocompatibility complex (MHC) proteins are involved in initiation of immune responses to foreign antigens via presentation of peptides to receptors of CD4(+) T-cells. An analogous presentation of self-peptides may lead to autoimmune diseases, such as rheumatoid arthritis (RA). The glycopeptide fragment CII259-273, derived from type II collagen, is presented by A(q) MHCII molecules in the mouse and has a key role in development of collagen induced arthritis (CIA), a validated model for RA. We have introduced hydroxyethylene amide bond isosteres at the Ala(261)-Gly(262) position of CII259-273. Biological evaluation showed that A(q) binding and T cell recognition were dramatically reduced for the modified glycopeptides, although static models predicted similar binding modes as the native type II collagen fragment. Molecular dynamics (MD) simulations demonstrated that introduction of the hydroxyethylene isosteres disturbed the entire hydrogen bond network between the glycopeptides and A(q). As a consequence the hydroxyethylene isosteric glycopeptides were prone to dissociation from A(q) and unfolding of the β1-helix. Thus, the isostere induced adjustment of the hydrogen bond network altered the structure and dynamics of A(q)/glycopeptide complexes leading to the loss of A(q) affinity and subsequent T cell response.

Major histocompatibility complex class II (MHCII) genes code for proteins that bind and present antigenic peptides and trigger the adaptive immune response. We present a broad geographical study of MHCII DA β1 (DAB) and DB β1 (DBB) variants of the koala (Phascolarctos cinereus; n=191) from 12 populations across eastern Australia, with a total of 13 DAB and 7 DBB variants found. We identified greater MHCII variation and, possibly, additional gene copies in koala populations in the north (Queensland and New South Wales) relative to the south (Victoria), confirmed by STRUCTURE analyses and genetic differentiation using analysis of molecular variance. The higher MHCII diversity in the north relative to south could potentially be attributed to (i) significant founder effect in Victorian populations linked to historical translocation of bottlenecked koala populations and (ii) increased pathogen-driven balancing selection and/or local genetic drift in the north. Low MHCII genetic diversity in koalas from the south could reduce their potential response to disease, although the three DAB variants found in the south had substantial sequence divergence between variants. This study assessing MHCII diversity in the koala with historical translocations in some populations contributes to understanding the effects of population translocations on functional genetic diversity.

The structural basis of the interaction between the CD4 coreceptor and a class II major histocompatibility complex (MHC) is described. The crystal structure of a complex containing the human CD4 N-terminal two-domain fragment and the murine I-A{sup k }class IIMHC molecule with associated peptide (pMHCII) shows that only the 'top corner' of the CD4 molecule directly contacts pMHCII. The CD4 Phe-43 side chain extends into a hydrophobic concavity formed by MHC residues from both {alpha}2 and {beta}2 domains. A ternary model of the CD4-pMHCII-T-cell receptor (TCR) reveals that the complex appears V-shaped with the membrane-proximal pMHCII at the apex. This configuration excludes a direct TCR-CD4 interaction and suggests how TCR and CD4 signaling is coordinated around the antigenic pMHCII complex. Human CD4 binds to HIV gp120 in a manner strikingly similar to the way in which CD4 interacts with pMHCII. Additional contacts between gp120 and CD4 give the CD4-gp120 complex a greater affinity. Thus, ligation of the viral envelope glycoprotein to CD4 occludes the pMHCII-binding site on CD4, contributing to immunodeficiency.

The standard model of Major Histocompatibility Complex class II (MHCII)-restricted antigen processing depicts a straightforward, linear pathway: internalized antigens are converted into peptides that load in a chaperone dependent manner onto nascent MHCII in the late endosome, the complexes subsequently trafficking to the cell surface for recognition by CD4+ T cells (TCD4+). Several variations on this theme, both moderate and radical, have come to light but these alternatives have remained peripheral, the conventional pathway generally presumed to be the primary driver of TCD4+ responses. Here we continue to press for the conceptual repositioning of these alternatives toward the center while proposing that MHCII processing be thought of less in terms of discrete pathways and more in terms of a network whose major and minor conduits are variable depending upon many factors, including the epitope, the nature of the antigen, the source of the antigen, and the identity of the antigen-presenting cell. PMID:24379819

The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation. PMID:9649539

The genomic sequences of 15 horse Major Histocompatibility Complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and non-classical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal, and two to three non-classical sequences. Phylogenetic analysis was applied to these sequences and groups were identified which appear to be allelic series, but some sequences were left ungrouped. Sequences determined from MHC class I heterozygous horses and previously described MHC class I sequences were then added, representing a total of ten horse MHC haplotypes. These results were consistent with those obtained from the MHC homozygous horses alone, and 30 classical sequences were assigned to four previously confirmed loci and three new provisional loci. The non-classical genes had few alleles and the classical genes had higher levels of allelic polymorphism. Alleles for two classical loci with the expected pattern of polymorphism were found in the majority of haplotypes tested, but alleles at two other commonly detected loci had more variation outside of the hypervariable region than within. Our data indicate that the equine Major Histocompatibility Complex is characterized by variation in the complement of class I genes expressed in different haplotypes in addition to the expected allelic polymorphism within loci. PMID:20099063

Nuclear Factor Y (NF-Y) was first described as one of the CCAAT binding factors. Although CCAAT motifs were found to be present in various genes, NF-Y attracted a lot of interest early on, due to its role in Major Histocompatibility Complex (MHC) gene regulation. MHC genes are crucial in immune response and show peculiar expression patterns. Among other conserved elements on MHC promoters, an NF-Y binding CCAAT box was found to contribute to MHC transcriptional regulation. NF-Y along with other DNA binding factors assembles in a stereospecific manner to form a multiprotein scaffold, the MHC enhanceosome, which is necessary but not sufficient to drive transcription. Transcriptional activation is achieved by the recruitment of yet another factor, the class II transcriptional activator (CIITA). In this review, we briefly discuss basic findings on MHCII transcription regulation and we highlight NF-Y different modes of function in MHCII gene activation.

Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class IIMHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-gamma. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.

Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4 + T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4 + T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

Major histocompatibility complex (MHC) genes play important role in host-parasite interactions and parasites are crucial factors influencing the population dynamics of hosts. We described the structure and diversity of exon 2 of the MHC class II DQA gene in three species of voles (Arvicolinae) exhibiting regular multi-annual fluctuations of population density and analysed the processes leading to the observed MHC polymorphism. By using cloning-sequencing methodology and capillary electrophoresis-single strand conformation polymorphism, we described seven sequences in the water, eight in the common, and seven in the bank voles coming from an area of 70 km(2) around the Nozeroy canton in the Jura Mountains (Franche Comté, France). All exon 2 sequences translate to give unique amino acid sequences and positive selection was found to act very intensively on antigen binding sites. We documented the presence of recombination at vole DQA region but its importance in generating allelic polymorphism seems to be relatively limited. For the first time within rodents, we documented the duplication of the DQA gene in all three species with both copies being transcriptionally active. Phylogenetic analysis of allelic sequences revealed extensive trans-species polymorphism within the subfamily although no alleles were shared between species in our data set. We discuss possible role of parasites in forming the recent polymorphism pattern of the DQA locus in voles.

Generalized vitiligo is a common autoimmune disease in which acquired patchy depigmentation of skin, hair, and mucous membranes results from loss of melanocytes from involved areas. Previous genetic analyses have focused on vitiligo susceptibility, and have identified a number of genes involved in disease risk. Age of onset of generalized vitiligo also involves a substantial genetic component, but has not previously been studied systematically. In this study, we report a genome-wide association study of vitiligo age of onset in 1,339 generalized vitiligo patients, with replication in an independent cohort of 677 cases. We identified a quantitative trait locus for vitiligo age of onset in the major histocompatibility complex (MHC) class II region, located near c6orf10-BTNL2 (rs7758128; P = 8.14×10−11), a region that is also associated with generalized vitiligo susceptibility. In contrast, there was no association of vitiligo age of onset with any other MHC or non-MHC loci that are associated with vitiligo susceptibility. These findings highlight the differing roles played by genes involved in vitiligo susceptibility versus vitiligo age of onset, and illustrate that genome-wide analyses can be used to identify genes involved in quantitative aspects of disease natural history, as well as disease susceptibility per se. PMID:21326295

Targeting of MHC class II molecules to the endocytic compartment where they encounter processed antigen is determined by the invariant chain (Ii). By analysis of Ii-transferrin receptor (TR) chimera trafficking, we have identified sorting signals in the Ii cytoplasmic tail and transmembrane region that mediate this process. Two non-tyrosine-based sorting signals in the Ii cytoplasmic tail were identified that mediate localization to plasma membrane clathrin-coated pits and promote rapid endocytosis. Leu7 and Ile8 were required for the activity of the signal most distal to the cell membrane whereas Pro15 Met16 Leu17 were important for the membrane-proximal signal. The same or overlapping non- tyrosine-based sorting signals are essential for delivery of Ii-TR chimeras, either by an intracellular route or via the plasma membrane, to an endocytic compartment where they are rapidly degraded. The Ii transmembrane region is also required for efficient delivery to this endocytic processing compartment and contains a signal distinct from the Ii cytoplasmic tail. More than 80% of the Ii-TR chimera containing the Ii cytoplasmic tail and transmembrane region is delivered directly to the endocytic pathway by an intracellular route, implying that the Ii sorting signals are efficiently recognized by sorting machinery located in the trans-Golgi. PMID:8034737

Background Natural killer (NK) cells are an important resource of the innate immune system directly involved in the spontaneous recognition and lysis of virus-infected and tumor cells. An exquisite balance of inhibitory and activating receptors tightly controls the NK cell activity. At present, one of the best-characterized activating receptors is NKG2D, which promotes the NK-mediated lysis of target cells by binding to a family of cell surface ligands encoded by the MHC class I chain-related (MIC) genes, among others. The goal of this study was to describe the expression pattern of MICA and MICB at the molecular and cellular levels in human cervical cancer cell lines infected or not with human papillomavirus, as well as in a non-tumorigenic keratinocyte cell line. Results Here we show that MICA and MICB exhibit differential expression patterns among HPV-infected (SiHa and HeLa) and non-infected cell lines (C33-A, a tumor cell line, and HaCaT, an immortalized keratinocyte cell line). Cell surface expression of MICA was higher than cell surface expression of MICB in the HPV-positive cell lines; in contrast, HPV-negative cells expressed lower levels of MICA. Interestingly, the MICA levels observed in C33-A cells were overcome by significantly higher MICB expression. Also, all cell lines released higher amounts of soluble MICB than of soluble MICA into the cell culture supernatant, although this was most pronounced in C33-A cells. Additionally, Real-Time PCR analysis demonstrated that MICA was strongly upregulated after genotoxic stress. Conclusions This study provides evidence that even when MICA and MICB share a high degree of homology at both genomic and protein levels, differential regulation of their expression and cell surface appearance might be occurring in cervical cancer-derived cells. PMID:21631944

The development of fluorochrome-conjugated peptide–major histocompatibility complex (pMHC) multimers in conjunction with continuing advances in flow cytometry has transformed the study of antigen-specific T cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we bring together and discuss some of the ‘tricks’ that can be used to get the most out of pMHC multimers. These include: (1) simple procedures that can substantially enhance the staining intensity of cognate T cells with pMHC multimers; (2) the use of pMHC multimers to stain T cells with very-low-affinity T-cell receptor (TCR)/pMHC interactions, such as those that typically predominate in tumour-specific responses; and (3) the physical grading and clonotypic dissection of antigen-specific T cells based on the affinity of their cognate TCR using mutant pMHC multimers in conjunction with new approaches to the molecular analysis of TCR gene expression. We also examine how soluble pMHC can be used to examine T-cell activation, manipulate T-cell responses and study allogeneic and superantigen interactions with TCRs. Finally, we discuss the problems that arise with pMHC class II (pMHCII) multimers because of the low affinity of TCR/pMHCII interactions and lack of ‘coreceptor help’. PMID:19125886

According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage.

According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage.

According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage. PMID:27143688

The stable binding of processed foreign peptide to a class II major histocompatibility (MHC) molecule and subsequent presentation to a T cell receptor is a central event in immune recognition and regulation. Polymorphic residues on the floor of the peptide binding site form pockets that anchor peptide side chains. These and other residues in the helical wall of the groove determine the specificity of each allele and define a motif. Allele specific motifs allow the prediction of epitopes from the sequence of pathogens. There are, however, known epitopes that do not satisfy these motifs: anchor motifs are not adequate for predicting epitopes as there are apparently major and minor motifs. We present crystallographic studies into the nature of the interactions that govern the binding of these so called nonconforming peptides. We would like to understand the role of the P10 pocket and find out whether the peptides that do not obey the consensus anchor motif bind in the canonical conformation observed in in prior structures of class IIMHC-peptide complexes. HLA-DRB3*0101 complexed with peptide crystallized in unit cell 92.10 x 92.10 x 248.30 (90, 90, 90), P41212, and the diffraction data is reliable to 2.2ÅWe are complementing our studies with dynamical long time simulations to answer these questions, particularly the interplay of the anchor motifs in peptide binding, the range of protein and ligand conformations, and water hydration structures.

The MHC class I chain–related molecules (MICs) have previously been shown to be induced on most epithelial tumor cells. Engagement of MIC by the activating immune receptor NKG2D triggers NK cells and augments antigen-specific CTL anti-tumor immunity. The MIC-NKG2D system was proposed to participate in epithelial tumor immune surveillance. Paradoxically, studies suggest that tumors may evade MIC-NKG2D–mediated immunity by MIC shedding–induced impairment of effector cell function. Here we demonstrate the first evidence to our knowledge of a significant correlation of MIC shedding and deficiency in NK cell function with the grade of disease in prostate cancer. MIC is widely expressed in prostate carcinoma. The presence of surface target MIC, however, is counteracted by shedding. A significant increase in serum levels of soluble MIC (sMIC) and deficiency in NK cell function was shown in patients with advanced cancer. Finally, the deficiency in NK cell function can be overcome by treatment with IL-2 or IL-15 in vitro. Our results suggest that (a) deficiency in MIC-NKG2D immune surveillance may contribute to prostate cancer progression, (b) sMIC may be a novel biomarker for prostate cancer, and (c) using cytokines to restore MIC-NKG2D–mediated immunity may have clinical significance for prostate cancer in cell-based adaptive immunotherapy. PMID:15314693

In some mating combinations in rats, there is a maternal antibody response to the maternal antigenic components of the placenta without any previous immunization of the mother. The highest response occurs in the WF (u) female mated to the DA (a) male, and it is against a unique MHC-encoded class I antigen, the Pa antigen, and not against the major allele-specific transplantation antigen of the DA strain, RT1.Aa. The development of mAbs to the Pa and Aa antigens allowed us to localize these antigens on the placenta and to explore the reason for the differential antibody response to them using immunohistochemical and biochemical techniques. Both antibodies reacted with the WF X DA placenta and stained the endovascular and interstitial trophoblast of the decidua, the basal trophoblast, Reichert's membrane, and the yolk sac epithelium, but they did not stain the labyrinthine trophoblast. Blocking studies showed that each antibody reacted with a separate molecule in the placenta. Anti-class II mAbs reactive with the a or u haplotype did not stain the WF X DA, DA X DA, or WF X WF placenta; hence, there are no class II antigens in the placenta. Electron microscopic studies of the semiallogeneic WF X DA placenta using the immunogold technique with both single- and double-labeling showed that only the Pa antigen was expressed on the surface of the basal trophoblast, but that both the Pa and Aa antigens were in the cytoplasm of these cells; neither antigen was found in the labyrinthine trophoblast. By contrast, the placenta from the syngeneic DA X DA mating expressed both the Pa and Aa antigens on the surface of the basal trophoblast as well as in the cytoplasm; neither antigen was found in the labyrinthine trophoblast. These observations were quantified morphometrically using electron photomicrographs of single- labeled tissues. Both the Pa and Aa antigens isolated from the plasma membrane of lymphocytes have heavy chains of 46 kD, but those antigens isolated from the

The development of neutralizing anti-drug-antibodies to the Factor VIII protein-therapeutic is currently the most significant impediment to the effective management of hemophilia A. Common non-synonymous single nucleotide polymorphisms (ns-SNPs) in the F8 gene occur as six haplotypes in the human population (denoted H1 to H6) of which H3 and H4 have been associated with an increased risk of developing anti-drug antibodies. There is evidence that CD4+ T-cell response is essential for the development of anti-drug antibodies and such a response requires the presentation of the peptides by the MHC-class-II (MHC-II) molecules of the patient. We measured the binding and half-life of peptide-MHC-II complexes using synthetic peptides from regions of the Factor VIII protein where ns-SNPs occur and showed that these wild type peptides form stable complexes with six common MHC-II alleles, representing 46.5% of the North American population. Next, we compared the affinities computed by NetMHCIIpan, a neural network-based algorithm for MHC-II peptide binding prediction, to the experimentally measured values and concluded that these are in good agreement (area under the ROC-curve of 0.778 to 0.972 for the six MHC-II variants). Using a computational binding predictor, we were able to expand our analysis to (a) include all wild type peptides spanning each polymorphic position; and (b) consider more MHC-II variants, thus allowing for a better estimation of the risk for clinical manifestation of anti-drug antibodies in the entire population (or a specific sub-population). Analysis of these computational data confirmed that peptides which have the wild type sequence at positions where the polymorphisms associated with haplotypes H3, H4 and H5 occur bind MHC-II proteins significantly more than a negative control. Taken together, the experimental and computational results suggest that wild type peptides from polymorphic regions of FVIII constitute potential T-cell epitopes and thus

The identification and validation of new cancer-specific T cell epitopes continues to be a major area of research interest. Nevertheless, challenges remain to develop strategies that can easily discover and validate epitopes expressed in primary cancer cells. Regarded as targets for T cells, peptides presented in the context of the major histocompatibility complex (MHC) are recognized by monoclonal antibodies (mAbs). These mAbs are of special importance as they lend themselves to the detection of epitopes expressed in primary tumor cells. Here, we use an approach that has been successfully utilized in two different infectious disease applications (WNV and influenza). A direct peptide-epitope discovery strategy involving mass spectrometric analysis led to the identification of peptide YLLPAIVHI in the context of MHC A*02 allele (YLL/A2) from human breast carcinoma cell lines. We then generated and characterized an anti-YLL/A2 mAb designated as RL6A TCRm. Subsequently, the TCRm mAb was used to directly validate YLL/A2 epitope expression in human breast cancer tissue, but not in normal control breast tissue. Moreover, mice implanted with human breast cancer cells grew tumors, yet when treated with RL6A TCRm showed a marked reduction in tumor size. These data demonstrate for the first time a coordinated direct discovery and validation strategy that identified a peptide/MHC complex on primary tumor cells for antibody targeting and provide a novel approach to cancer immunotherapy.

Potent and broad cellular immune responses against the nonstructural (NS) proteins of hepatitis C virus (HCV) are associated with spontaneous viral clearance. In this study, we have improved the immunogenicity of an adenovirus (Ad)-based HCV vaccine by fusing NS3 from HCV (Strain J4; Genotype 1b) to the MHC class II chaperone protein invariant chain (Ii). We found that, after a single vaccination of C57BL/6 or BALB/c mice with Ad-IiNS3, the HCV NS3-specific CD8(+) T cell responses were significantly enhanced, accelerated, and prolonged compared with the vaccine encoding NS3 alone. The AdIiNS3 vaccination induced polyfunctional CD8(+) T cells characterized by coproduction of IFN-γ, TNF-α and IL-2, and this cell phenotype is associated with good viral control. The memory CD8(+) T cells also expressed high levels of CD27 and CD127, which are markers of long-term survival and maintenance of T cell memory. Functionally, the AdIiNS3-vaccinated mice had a significantly increased cytotoxic capacity compared with the AdNS3 group. The AdIiNS3-induced CD8(+) T cells protected mice from infection with recombinant vaccinia virus expressing HCV NS3 of heterologous 1b strains, and studies in knockout mice demonstrated that this protection was mediated primarily through IFN-γ production. On the basis of these promising results, we suggest that this vaccination technology should be evaluated further in the chimpanzee HCV challenge model.

Immune responses to HLA and development of anti-donor HLA (DSA) have been shown to play a role in chronic rejection following transplantation. We hypothesized that Abs to MHC changes microRNAs (miRNAs) leading to chronic lung allograft rejection. Microarray analysis was performed in a murine model of anti-MHC induced obliterative airway disease (OAD), a correlate of obliterative bronchiolitis. A unique profile of dysregulated miRNAs was detected in OAD mice on day 7 and 15 after Ab administration compared to control. Sixty-seven miRNAs were increased and 42 miRNAs were decreased in OAD mice on day 7. In addition, 15 miRNAs were over expressed and 16 miRNAs were under expressed in OAD mice on day 15. The expression of miR-16 and miR-195 were significantly decreased in lungs of OAD mice by qPCR and in situ hybridization with increases of H-2 Aa and H-2 Dma mRNA levels. Significant reduction of miR-16 and miR-195 levels were also noted in lung transplant (LTx) patients with DSA compared to LTx without DSA. Bioinformatic TargetScan and Reporter Assays identified the binding of miR-16 and miR-195 to the 3’UTR of Regulatory Factor X 5. qPCR and immunohistochemistry indicated post-transcriptional increases of Regulatory Factor X 5 mRNA and protein expression not only in OAD mice and but also in LTxR with DSA which was associated with increased expression of HLA-DPA1, HLA-DQA1, and HLA-DRA mRNA. Therefore, our results demonstrated that miRNAs induced by alloimmunity may play important roles in chronic rejection after LTx. PMID:25941328

The repertoire of peptides displayed in vivo by MHCII molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHCII peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin β4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHCII self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of “self-recognition” as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis. PMID:26740625

A panel of monoclonal antibodies to monomorphic determinants of the MHC class II subregion locus products: DP, DR and DQ, was used to investigate the expression of these antigens on early lymphocytes and macrophages from human fetal liver (13-20 weeks), placenta (16 weeks and term) and cord blood, in relation to the class II phenotype of cells from adult tonsil and peripheral blood. Fetal liver sections and cell suspensions showed differential expression of class II antigens. DP was expressed at a higher frequency (11.0% of nucleated cells) than DR on lymphoid cells and macrophages from fetal liver, and DQ was either absent or expressed on less than 0.3% of nucleated cells. Consistent with this finding, DP but not DR or DQ antigens were observed on vascular elements and macrophages in the villi of 16-week placenta. At term, all three subregion locus products were expressed. Adult tonsil and peripheral blood B lymphocytes expressed DP, DR and DQ antigens with similar frequency; however, DQ was expressed at a lower frequency than DP and DR on cord blood B lymphocytes. In contrast, 30-50% macrophages from cord blood and adult peripheral blood expressed DP and DR, but fewer (5% and 18%, respectively) expressed DQ. These data suggest that class II antigens are expressed in the sequence DP, DR, DQ on developing lymphocytes. A similar sequence is suggested for macrophages. Images Figure 1 Figure 2 Figure 3 PMID:3894221

Systemic juvenile idiopathic arthritis (sJIA) is an often severe, potentially life-threatening childhood inflammatory disease, the pathophysiology of which is poorly understood. To determine whether genetic variation within the MHC locus on chromosome 6 influences sJIA susceptibility, we performed an association study of 982 children with sJIA and 8,010 healthy control subjects from nine countries. Using meta-analysis of directly observed and imputed SNP genotypes and imputed classic HLA types, we identified the MHC locus as a bona fide susceptibility locus with effects on sJIA risk that transcended geographically defined strata. The strongest sJIA-associated SNP, rs151043342 [P = 2.8 × 10(-17), odds ratio (OR) 2.6 (2.1, 3.3)], was part of a cluster of 482 sJIA-associated SNPs that spanned a 400-kb region and included the class II HLA region. Conditional analysis controlling for the effect of rs151043342 found that rs12722051 independently influenced sJIA risk [P = 1.0 × 10(-5), OR 0.7 (0.6, 0.8)]. Meta-analysis of imputed classic HLA-type associations in six study populations of Western European ancestry revealed that HLA-DRB1*11 and its defining amino acid residue, glutamate 58, were strongly associated with sJIA [P = 2.7 × 10(-16), OR 2.3 (1.9, 2.8)], as was the HLA-DRB1*11-HLA-DQA1*05-HLA-DQB1*03 haplotype [6.4 × 10(-17), OR 2.3 (1.9, 2.9)]. By examining the MHC locus in the largest collection of sJIA patients assembled to date, this study solidifies the relationship between the class II HLA region and sJIA, implicating adaptive immune molecules in the pathogenesis of sJIA.

Leishmaniasis is a significant worldwide health problem for which no vaccine exists. Activation of CD4(+) and CD8(+) T cells is crucial for the generation of protective immunity against parasite. Recent trend in vaccine design has been shifted to epitope-based vaccines that are more specific, safe, and easy to produce. In the present study, four known antigenic Leishmania infantum proteins, cysteine peptidase A (CPA), histone H1, KMP-11, and Leishmania eukaryotic initiation factor (LeIF) were analyzed for the prediction of binding epitopes to H2(d) MHC class I and II molecules, using online available algorithms. Based on in silico analysis, eight peptides including highly scored MHC class I- and II-restricted epitopes were synthesized. Peptide immunogenicity was validated in MHC compatible BALB/c mice immunized with each synthetic peptide emulsified in complete Freund's adjuvant/incomplete Freund's adjuvant. CPA_p2, CPA_p3, H1_p1, and LeIF_p6 induced strong spleen cell proliferation upon in vitro peptide re-stimulation. In addition, the majority of the peptides, except of LeIF_p1 and KMP-11_p1, induced IFN-γ secretion, while KMP-11_p1 indicated a suppressive effect on IL-10 production. CPA_p2, CPA_p3, LeIF_p3, and LeIF_p6 induced IFN-γ-producing CD4(+) T cells indicating a TH1-type response. In addition, CPA_p2, CPA_p3, and H1_p1 induced also the induction of CD8(+) T cells. The induction of peptide-specific IgG in immunized mice designated also the existence of B cell epitopes in peptide sequences. Combining immunoinformatic tools and experimental validation, we demonstrated that CPA_p2, CPA_p3, H1_p1, H1_p3, CPA_p2, LeIF_p3, and LeIF_p6 are likely to include potential epitopes for the induction of protective cytotoxic and/or TH1-type immune responses supporting the feasibility of peptide-based vaccine development for leishmaniasis.

Polyclonality of self-reactive CD4(+) T cells is the hallmark of several autoimmune diseases like type 1 diabetes. We have previously reported that a soluble dimeric MHCII-peptide chimera prevents and reverses type 1 diabetes induced by a monoclonal diabetogenic T-cell population in double Tg mice [Casares, S. et al., Nat. Immunol. 2002. 3: 383-391]. Since most of the glutamic acid decarboxylase 65 (GAD65)-specific CD4(+) T cells in the NOD mouse are tolerogenic but unable to function in an autoimmune environment, we have activated a silent, monoclonal T-regulatory cell population (GAD65(217-230)-specific CD4(+) T cells) using a soluble I-A(αβ) (g7)/GAD65(217-230)/Fcγ2a dimer, and measured the effect on the ongoing polyclonal diabetogenic T-cell process. Activated GAD65(217-230)-specific T cells and a fraction of the diabetogenic (B(9-23)-specific) T cells were polarized toward the IL-10-secreting T-regulatory type 1-like function in the pancreas of diabetic NOD mice. More importantly, this led to the reversal of hyperglycemia for more than 2 months post-therapy in 80% of mice in the context of stabilization of pancreatic insulitis and improved insulin secretion by the β cells. These findings argue for the stabilization of a polyclonal self-reactive T-cell process by a single epitope-mediated bystander suppression. Dimeric MHC class II-peptide chimeras-like approach may provide rational grounds for the development of more efficient antigen-specific therapies in type 1 diabetes.

Beta 2-microglobulin (beta 2m) forms the invariant light chain of the MHC-encoded HLA-ABC and the non-MHC-encoded CD1 molecules. While HLA-ABC (MHC Class I) molecules are virtually ubiquitous in tissue distribution, CD1 determinants by contrast are more restricted. We have assessed, by indirect immunoenzymeassay, the relative membrane densities of these molecules on malignant thymic and post-thymic T cells. It was found that the T cells of mature post-thymic proliferations expressed significantly more beta 2m-associated protein, predominantly HLA-ABC in nature, than thymic-ALL blasts. This parallels the situation found in normal peripheral T cells and thymocytes. In contrast to post-thymic T cells, thymic-ALL blasts showed considerable case to case variation with respect to non-HLA-associated beta 2m and, of particular interest, not all of this excess beta 2m could be accounted for by CD1a. We therefore conclude that other beta 2m-containing molecules may be expressed on thymic-ALL blasts and possibly also on post-thymic leukaemic T cells. In addition, it was found that T cells from CD4+ cases of post-thymic proliferations expressed more beta 2m-associated determinants than other T cells, whether of either normal or malignant origin, and that certain post-thymic malignancies express significantly increased levels of beta 2m-associated protein relative to normal peripheral T-cells. This is in direct contrast to the situation seen in many solid malignancies. PMID:2466592

Most T cells have T cell receptors (TCR) of micromolar affinity for peptide-major histocompatibility complex (MHC) ligands, but genetic engineering can generate TCRs of nanomolar affinity. The affinity of the TCR used, m33, for its cognate non-self peptide–MHC-I complex (SIYRYYGL-Kb) is 1,000-fold higher than of the wild-type TCR 2C. The affinity of m33 for the self-peptide dEV-8 on Kb is only twofold higher. Mouse CD8+ T cells transduced with an m33-encoding retrovirus showed binding of SIY-Kb and potent function in vitro, but in vivo these T cells disappeared within hours after transfer into syngeneic hosts without causing graft-versus-host disease (GVHD). Accordingly, in cases where such CD8-dependent self-reactivity might occur in human adoptive T cell therapies, our results show that a peripheral T-cell deletion mechanism could operate to avoid reactions with the host. In contrast to CD8+ T cells, we show that CD4+ T cells expressing m33 survived for months in vivo. Furthermore, the m33-transduced CD4+ T cells were able to mediate antigen-specific rejection of 6-day-old tumors. Together, we show that CD8+ T cell expressing a MHC class I-restricted high-affinity TCR were rapidly deleted whereas CD4+ T cells expressing the same TCR survived and provided function while being directed against a class I-restricted antigen. PMID:22233579

We sequenced exon 2 of the major histocompatibility complex (MHC) class II DRB3 gene from 471 individuals in four different Japanese populations of cattle (201 Japanese Black, 101 Holstein, 100 Japanese Shorthorn, and 69 Jersey cattle) using a new method for sequence-based typing (SBT). We identified the 34 previously reported alleles and four novel alleles. These alleles were 80.0-100.0% identical at the nucleotide level and 77.9-100.0% identical at the amino acid level to the bovine MHC (BoLA)-DRB3 cDNA clone NR1. Among the 38 alleles, eight alleles were found in only one breed in this study. However, these alleles did not form specific clusters on a phylogenetic tree of 236-base pairs (bp) nucleotide sequences. Furthermore, these breeds exhibited similar variations with respect to average frequencies of nucleotides and amino acids, as well as synonymous and non-synonymous substitutions, in all pairwise comparisons of the alleles found in this study. By contrast, analysis of the frequencies of the various BoLA-DRB3 alleles in each breed indicated that DRB3*1101 was the most frequent allele in Holstein cattle (16.8%), DRB3*4501 was the most frequent allele in Jersey cattle (18.1%), DRB3*1201 was the most frequent allele in Japanese Shorthorn cattle (16.0%) and DRB3*1001 was the most frequent allele in Japanese Black cattle (17.4%), indicating that the frequencies of alleles were differed in each breed. In addition, a population tree based on the frequency of BoLA-DRB3 alleles in each breed suggested that Holstein and Japanese Black cattle were the most closely related, and that Jersey cattle were more different from both these breeds than Japanese Shorthorns.

Genetic diversity is one of the pillars of conservation biology research. High genetic diversity and abundant genetic variation in an organism may be suggestive of capacity to adapt to various environmental changes. The major histocompatibility complex (MHC) is known to be highly polymorphic and plays an important role in immune function. It is also considered an ideal model system to investigate genetic diversity in wildlife populations. The Rufous-backed Bunting (Emberiza jankowskii) is an endangered species that has experienced a sharp decline in both population and habitat size. Many historically significant populations are no longer present in previously populated regions, with only three breeding populations present in Inner Mongolia (i.e., the Aolunhua, Gahaitu and Lubei557 populations). Efforts focused on facilitating the conservation of the Rufous-backed Bunting (Emberiza jankowskii) are becoming increasingly important. However, the genetic diversity of E. jankowskii has not been investigated. In the present study, polymorphism in exon 2 of the MHCIIB of E. jankowskii was investigated. This polymorphism was subsequently compared with a related species, the Meadow Bunting (Emberiza cioides). A total of 1.59 alleles/individual were detected in E. jankowskii and 1.73 alleles/individual were identified in E. cioides. The maximum number of alleles per individual from the three E. jankowskii populations suggest the existence of at least three functional loci, while the maximum number of alleles per individual from the three E. cioides populations suggest the presence of at least four functional loci. Two of the alleles were shared between the E. jankowskii and E. cioides. Among the 12 unique alleles identified in E. jankowskii, 10.17 segregating sites per allele were detected, and the nucleotide diversity was 0.1865. Among the 17 unique alleles identified in E. cioides, eight segregating sites per allele were detected, and the nucleotide diversity was 0

The major histocompatibility complex (MHC) is an excellent molecular marker for the studies of evolutionary ecology and conservation genetics because it is a family of highly polymorphic genes that play a key role in vertebrate immune response. In this study, the functional genes of MHC Class II B (DAB) were isolated for the first time in a vulnerable species, the Chinese egret (Egrettaeulophotes). Using a full length DNA and cDNA produced by PCR and RACE methods, four potential MHC DAB loci were characterized in the genome of this egret and all four were expressed in liver and blood. At least four copies of the MHC gene complex were similar to two copies of the minimal essential MHC complex of chicken, but are less complex than the multiple copies expressed in passerine species. In MHC polymorphism, 19 alleles of exon 2 were isolated from 48 individuals using PCR. No stop codons or frameshift mutations were found in any of the coding regions. The signatures of positive selection detected in potential peptide-binding regions by Bayesian analysis, suggesting that all of these genes were functional. These data will provide the fundamental basis for further studies to elucidate the mechanisms and significance of MHC molecular adaptation in vulnerable Chinese egret and other ardeids.

Professional antigen-presenting cells (APCs) such as conventional dendritic cells (DCs) process protein antigens to MHC-bound peptides and then present the peptide–MHC complexes to T cells. In addition to this canonical antigen presentation pathway, recent studies have revealed that DCs and non-APCs can acquire MHC class I (MHCI) and/or MHC class II (MHCII) from neighboring cells through a process of cell–cell contact-dependent membrane transfer called trogocytosis. These MHC-dressed cells subsequently activate or regulate T cells via the preformed antigen peptide–MHC complexes without requiring any further processing. In addition to trogocytosis, intercellular transfer of MHCI and MHCII can be mediated by secretion of membrane vesicles such as exosomes from APCs, generating MHC-dressed cells. This review focuses on the physiological role of antigen presentation by MHCI- or MHCII-dressed cells, and also discusses differences and similarities between trogocytosis and exosome-mediated transfer of MHC. PMID:25601867

The role of thymus-independent type 2 (TI-2) antigens (polysaccharides) on the MHC-II-restricted processing of protein antigens was studied in vitro. In general, antigen presentation is inhibited when both peritoneal and splenic macrophages (M phi) as well as Küpffer cells (KC) are preincubated with acidic polysaccharides or branched dextrans. However, the inhibitory effect of neutral polysaccharides was minimal when KC were used as antigen presenting cells (APC). Morphological evaluation of the uptake of fluoresceinated polysaccharides clearly correlates with this selective and differential interference. Polysaccharides do not block MHC-I-restricted antigen presentation. Some chemical characteristics shared by different saccharides seem to be specially related to their potential inhibitory abilities: (i) those where two anomeric carbon atoms of two interlinked sugars and (ii) those containing several sulfate groups per disaccharide repeating unit. No polysaccharide being inhibitory in M phi abrogated antigen processing in other APC: lipopolysaccharide-activated B cells, B lymphoma cells, or dendritic cells (DC). Using radiolabeled polysaccharides it was observed that DC and B cells incorporated less radioactivity as a function of time than M phi. Morphological evaluation of these different APC incubated for extended periods of time with inhibitory concentrations of polysaccharides revealed intense cytoplasmic vacuolization in M phi but not in B cells or DC. The large majority of M phi lysosomes containing polysaccharides fail to fuse with incoming endocytic vesicles and delivery of fluid-phase tracers was reduced, suggesting that indigestible carbohydrates reduced the fusion of these loaded lysosomes with endosomes containing recently internalized tracers. It is suggested that the main causes of this antigen presentation blockade are (i) the chemical characteristics of certain carbohydrates and whether the specific enzymatic machinery for their intracellular

Major histocompatibility complex (Mhc) genes are frequently used as a model for adaptive genetic diversity. Although associations between Mhc and disease resistance are frequently documented, little is known about the fitness consequences of Mhc variation in wild populations. Further, most work to date has involved testing associations between Mhc genotypes and fitness components. However, the functional diversity of the Mhc, and hence the mechanism by which selection on Mhc acts, depends on how genotypes map to the functional properties of Mhc molecules. Here, we test three hypotheses that relate Mhc diversity to fitness: (i) the maximal diversity hypothesis, (ii) the optimal diversity hypothesis and (iii) effect of specific Mhc types. We combine mark-recapture methods with analysis of long-term breeding data to investigate the effects of Mhc class I functional diversity (Mhc supertypes) on individual fitness in a wild great tit (Parus major) population. We found that the presence of three different Mhc supertypes was associated with three different components of individual fitness: survival, annual recruitment and lifetime reproductive success (LRS). Great tits possessing Mhc supertype 3 experienced higher survival rates than those that did not, whereas individuals with Mhc supertype 6 experienced higher LRS and were more likely to recruit offspring each year. Conversely, great tits that possessed Mhc supertype 5 had reduced LRS. We found no evidence for a selective advantage of Mhc diversity, in terms of either maximal or optimal supertype diversity. Our results support the suggestion that specific Mhc types are an important determinant of individual fitness.

An experiment was carried out to study the preventive effect of bacterial meal (BM) produced from natural gas against plant-induced enteropathy in Atlantic salmon (Salmo salar). Salmon were fed a diet based on fish meal (FM) or seven diets with 200 g/kg solvent-extracted soyabean meal (SBM) to induce enteritis in combination with increasing levels of BM from 0 to 300 g/kg. Salmon fed a SBM-containing diet without BM developed typical SBM-induced enteritis. The enteritis gradually disappeared with increasing inclusion of BM. By morphometry, no significant (P>0.05) differences in the size of stretches stained for proliferating cell nuclear antigen were found with 150 g/kg BM compared with the FM diet. Increasing BM inclusion caused a gradual decline in the number of cluster of differentiation 8 α positive (CD8α+) intraepithelial lymphocytes, and fish fed BM at 200 g/kg or higher revealed no significant difference from the FM diet. Histological sections stained with antibody for MHC class II (MHCII) showed that fish with intestinal inflammation had more MHCII-reactive cells in the lamina propria and submucosa, but less in the epithelium and brush border, compared with fish without inflammation. There were no significant (P>0.05) differences in growth among the diets, but the highest levels of BM slightly reduced protein digestibility and increased the weight of the distal intestine. In conclusion, the prevention of SBM-induced enteritis by BM is dose dependent and related to intestinal levels of MHCII- and CD8α-reactive cells.

Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3-5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.

patients, and may provide a powerful tool for activation of the immune system against primary tumor and metastatic disease . Body: Statement of...cells with IL-12 reduces established metastatic disease and stimulates immune effectors and monokine-induced by interferon-gamma. Canc. Immunol...concomitant down-regulation of Ii via RNAi may further improve vaccine efficacy and protect and/or treat tumor recurrence and/ or metastatic disease

The avian MHC-associated gene set includes orthologs to genes found throughout the human major histocompatibility complex (MHC), including some loci of the evolutionarily conserved class III region. In the turkey and other Galliformes, genes linked to the MHC have been identified because they are closely associated with class I or class II genes. This study was designed to evaluate additional class III genes for linkage to the avian MHC to further determine conservation of these loci in birds. BLAST searches were used to locate sequences in the turkey genome with similarity to genes shared between the MHC of Xenopus and humans. Primers were designed to target 25 genes, and putative orthologs were amplified by PCR and sequenced. Sequence polymorphisms were identified for 15 genes in turkey reference mapping families, and 8 genes showed significant genetic linkage to the turkey MHC-B locus. These new genetic markers and linkage relationships broaden our understanding of the composition of the avian MHC and expand the gene content for the turkey MHC-B.

Rhesus macaques have long been used in animal models for various human diseases, the susceptibility and/or resistance to some of which have been associated with the major histocompatibilty complex (MHC). To gain insight into the MHC background and to facilitate the experimental use of Chinese rhesus macaques, the second exon of MhcMamu-DQB1 genes in 105 rhesus macaques were characterized by cloning and sequencing. A total of 37 MhcMamu-DQB1 alleles were identified, illustrating a marked allelic polymorphism at DQB1 in these monkeys. In addition to 10 alleles were novel sequences that had not been documented in earlier reports, at least 14 alleles reported in earlier studies were not detected in this study. Most of the sequences (73%) observed in this study belong to DQB1 06 (13 alleles) and DQB1 18 (14 alleles) lineages, and the rest (27%) belong to DQB1 15, DQB1 16 and DQB1 17 lineages. The most frequent allele detected among these monkeys was MhcMamu-DQB1 06111 (22%), followed by DQB1 1503 (19%); and most of the novel alleles were present at a frequency of less than 2.5%. As for individual animals, 24 of 105 (23%) were homozygous whereas 81 of 105 (77%) were heterozygous at the MhcMamu-DQB1 locus. These data indicated significant differences in MhcMamu-DQB1 allele distribution between the Chinese rhesus macaques and the previously reported rhesus macaques, which were mostly of Indian origin. This information will not only promote the understanding of rhesus macaque MHC diversity and polymorphism but will also facilitate the use of Chinese rhesus macaques in human disease studies, especially those that may be associated with HLA-DQB genes.

Little is known about the major histocompatibility complex (MHC) in the genome of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) (YFP) or other cetaceans. In this study, a high-quality YFP bacterial artificial chromosome (BAC) library was constructed. We then determined the organization and characterization of YFP MHC class II region by screening the BAC library, followed by sequencing and assembly of positive BAC clones. The YFP MHC class II region consists of two segregated contigs (218,725 bp and 328,435 bp respectively) that include only eight expressedMHC class II genes, three pseudo MHC genes and twelve non-MHC genes. The YFP has fewer MHC class II genes than ruminants, showing locus reduction in DRB, DQA, DQB, and loss of DY. In addition, phylogenic and evolutionary analyses indicated that the DRB, DQA and DQB genes might have undergone birth-and-death evolution, whereas the DQB gene might have evolved under positive selection in cetaceans. These findings provide an essential foundation for future work, such as estimating MHC genetic variation in the YFP or other cetaceans. This work is the first report on the MHC class II region in cetaceans and offers valuable information for understanding the evolution of MHC genome in cetaceans. PMID:26932528

Conventional MHC class Ia-restricted CD8(+) T cells play a dominant role in the host response to virus infections, but recent studies indicate that T cells with specificity for nonclassical MHC class Ib molecules may also participate in host defense. To investigate the potential role of class Ib molecules in anti-viral immune responses, K(b-/-)D(b-/-)CIITA(-/-) mice lacking expression of MHC class Ia and class II molecules were infected with lymphocytic choriomeningitis virus (LCMV). These animals have a large class Ib-selected CD8(+) T cell population and they were observed to mediate partial (but incomplete) virus clearance during acute LCMV infection as compared with K(b-/-)D(b-/-)β(2)-microglobulin(-/-) mice that lack expression of both MHC class Ia and class Ib molecules. Infection was associated with expansion of splenic CD8(+) T cells and induction of granzyme B and IFN-γ effector molecules in CD8(+) T cells. Partial virus clearance was dependent on CD8(+) cells. In vitro T cell restimulation assays demonstrated induction of a population of β(2)-microglobulin-dependent, MHC class Ib-restricted CD8(+) T cells with specificity for viral Ags and yet to be defined nonclassical MHC molecules. MHC class Ib-restricted CD8(+) T cell responses were also observed after infection of K(b-/-)D(b-/-)mice despite the low number of CD8(+) T cells in these animals. Long-term infection studies demonstrated chronic infection and gradual depletion of CD8(+) T cells in K(b-/-)D(b-/-)CIITA(-/-) mice, demonstrating that class Ia molecules are required for viral clearance. These findings demonstrate that class Ib-restricted CD8(+) T cells have the potential to participate in the host immune response to LCMV.

Changes were examined in the expression of Class I and II major histocompatibility complex (MHC) antigens by autochthonous cells of the terminal ileum affected by Crohn's disease. The study was based on the analysis of transmural specimens from terminal ileum segments obtained in the course of ileocolectomy for colon cancer and Crohn's disease. Serial sections were immunostained using monoclonal antibodies directed against monomorphic determinants of HLA-A,B,C, DR, DP, DQ, and the invariant chain (Ii) associated with Class II molecules. Compared with the normal state, the only change in Class I antigen expression occurring in Crohn's disease was the induction of HLA-A,B,C antigens in lymphatic endothelium. Changes in Class II antigen expression were more substantial. Enhancement of HLA-DR expression was found in enterocytes; DR induction was observed in glial cells of the visceral nervous plexus and in venular and venous endothelium. HLA-DP and DQ antigens were induced in enterocytes, glial cells, and capillary and venular endothelium, although this induction was restricted to areas of moderate or high inflammatory activity. The tissue distribution of Ii closely resembled that of HLA-DR, although this association was not strict: on the one hand, arterial endothelium contained low amounts of Ii in the absence of DR antigens; on the other hand, glial cells expressed Class II molecules in the absence of Ii. The extent of local enhancement/induction of MHC antigens was positively correlated with the local density of the cellular infiltrate. These data suggest that altered MHC antigen expression by autochthonous structures might be mediated by factors released from the lymphohistiocytic infiltrate, which is itself attracted by an unknown signal. In conjunction with an unknown antigen, the enhanced expression of Class II antigens might trigger an autoaggressive immune response. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3425689

The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus–transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1–2 μm), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules. PMID:9606205

The Chediak-Higashi syndrome (CHS) is a human recessive autosomal disease caused by mutations in a single gene encoding a protein of unknown function, called lysosomal-trafficking regulator. All cells in CHS patients bear enlarged lysosomes. In addition, T- and natural killer cell cytotoxicity is defective in these patients, causing severe immunodeficiencies. We have analyzed major histocompatibility complex class II functions and intracellular transport in Epstein Barr Virus-transformed B cells from CHS patients. Peptide loading onto major histocompatibility complex class II molecules and antigen presentation are strongly delayed these cells. A detailed electron microscopy analysis of endocytic compartments revealed that only lysosomal multilaminar compartments are enlarged (reaching 1-2 micron), whereas late multivesicular endosomes have normal size and morphology. In contrast to giant multilaminar compartments that bear most of the usual lysosomal markers in these cells (HLA-DR, HLA-DM, Lamp-1, CD63, etc.), multivesicular late endosomes displayed reduced levels of all these molecules, suggesting a defect in transport from the trans-Golgi network and/or early endosomes into late multivesicular endosomes. Further insight into a possible mechanism of this transport defect came from immunolocalizing the lysosomal trafficking regulator protein, as antibodies directed to a peptide from its COOH terminal domain decorated punctated structures partially aligned along microtubules. These results suggest that the product of the Lyst gene is required for sorting endosomal resident proteins into late multivesicular endosomes by a mechanism involving microtubules.

A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population. PMID:9824493

A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.

MHC haplotypes of humans and the African great ape species have one copy of the MHC-A, -B, and -C genes. In contrast, MHC haplotypes of orangutans, the Asian great ape species, exhibit variation in the number of gene copies. An in-depth analysis of the MHC class I gene repertoire in the two orangutan species, Pongo abelii and Pongo pygmaeus, is presented here. This analysis involved Sanger and next-generation sequencing methodologies, revealing diverse and complicated transcription profiles for orangutan MHC-A, -B, and -C. Thirty-five previously unreported MHC class I alleles are described. The data demonstrate that each orangutan MHC haplotype has one copy of the MHC-A gene, and that the MHC-B region has been subject to duplication, giving rise to at least three MHC-B genes. The MHC-B*03 and -B*08 lineages of alleles each account for a separate MHC-B gene. All MHC-B*08 allotypes have the C1-epitope motif recognized by KIR. At least one other MHC-B gene is present, pointing to MHC-B alleles that are not B*03 or B*08. The MHC-C gene is present only on some haplotypes, and each MHC-C allotype has the C1-epitope. The transcription profiles demonstrate that MHC-A alleles are highly transcribed, whereas MHC-C alleles, when present, are transcribed at very low levels. The MHC-B alleles are transcribed to a variable extent and over a wide range. For those orangutan MHC class I allotypes that are detected by human monoclonal anti-HLA class I antibodies, the level of cell-surface expression of proteins correlates with the level of transcription of the allele. PMID:26685209

Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3′-UTR, but not to the mutated 3′-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHCexpression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity. PMID:24728149

The association of primary Sjögren's syndrome (pSS) with Major Histocompatibility Complex (MHC) alleles is quintessential of MHC-disease associations. Indeed, although disease associations with classical HLA class I and II alleles/haplotypes are amply documented, further dissection is often prevented by the strong linkage disequilibrium across the entire MHC complex. Here we study the association of pSS, not with HLA genes, but with the non-conventional MHC encoded class I gene, MICA (MHC class I chain-related gene A). MICA is selectively expressed within epithelia, and is the major ligand for the activatory receptor, NKG2D, both attributes relevant to pSS' etiology. MICA-pSS association was studied in two independent (French and UK) cohorts representing a total of 959 cases and 1,043 controls. MICA*008 allele was shown to be significantly associated with pSS (pcor=2.61x10-35). A multivariate logistic regression showed that this association was independent of all major known MHC-linked risk loci/alleles, as well as other relevant candidate loci that are in linkage disequilibrium with MICA*008 i.e. HLA-B*08:01, rs3131619 (T), MICB*008, TNF308A, HLA-DRB1*03:01 and HLA-DRB1*15:01 (p=1.84x10-04). Furthermore, independently of the MICA*008 allele, higher levels of soluble MICA proteins were detected in sera of pSS patients compared to healthy controls. This study hence defines MICA as a new, MHC-linked, yet HLA-independent, pSS risk locus and opens a new front in our understanding of the still enigmatic pathophysiology of this disease. The fact that the soluble MICA protein is further amplified in MICA*008 carrying individuals, might also be relevant in other auto-immune diseases and cancer.

The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2–6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs. PMID:25503521

The major histocompatibility complex (MHC) is a dynamic genome region with an essential role in the adaptive immunity of vertebrates, especially antigen presentation. The MHC is generally divided into subregions (classes I, II and III) containing genes of similar function across species, but with different gene number and organisation. Crocodylia (crocodilians) are widely distributed and represent an evolutionary distinct group among higher vertebrates, but the genomic organisation of MHC within this lineage has been largely unexplored. Here, we studied the MHC region of the saltwater crocodile (Crocodylus porosus) and compared it with that of other taxa. We characterised genomic clusters encompassing MHC class I and class II genes in the saltwater crocodile based on sequencing of bacterial artificial chromosomes. Six gene clusters spanning ∼452 kb were identified to contain nine MHC class I genes, six MHC class II genes, three TAP genes, and a TRIM gene. These MHC class I and class II genes were in separate scaffold regions and were greater in length (2-6 times longer) than their counterparts in well-studied fowl B loci, suggesting that the compaction of avian MHC occurred after the crocodilian-avian split. Comparative analyses between the saltwater crocodile MHC and that from the alligator and gharial showed large syntenic areas (>80% identity) with similar gene order. Comparisons with other vertebrates showed that the saltwater crocodile had MHC class I genes located along with TAP, consistent with birds studied. Linkage between MHC class I and TRIM39 observed in the saltwater crocodile resembled MHC in eutherians compared, but absent in avian MHC, suggesting that the saltwater crocodile MHC appears to have gene organisation intermediate between these two lineages. These observations suggest that the structure of the saltwater crocodile MHC, and other crocodilians, can help determine the MHC that was present in the ancestors of archosaurs.

Some members of the major histocompatibility complex (MHC) class I gene family are encoded outside the MHC. Here we describe a family of mouse class I-like genes mapping to the vicinity of the leukocyte receptor complex (LRC) on chromosome 7. This family, which we call Mill (MHC class I-like located near the LRC), has two members designated Mill1 and Mill2. Both genes are predicted to encode membrane glycoproteins with domain organization essentially similar to that of MHC class I heavy chains. The following features of Mill are noteworthy. (i) The deduced MILL proteins lack most of the residues known to be involved in the docking of peptides in classical MHC class I molecules. (ii) Among the known members of the class I gene family, MILL1 and MILL2 are related most closely to MICA/MICB encoded in the human MHC. (iii) Unlike all other known members of the class I gene family, Mill1 and Mill2 have an exon between those coding for the signal peptide and the α1 domain. (iv) Mill1 has a more restricted expression profile than Mill2. (v) The gene orthologous to Mill1 or Mill2 apparently is absent in the human. (vi) Mill1 and Mill2 show a limited degree of polymorphism in laboratory mice. The observation that the Mill family is related most closely to the MIC family, together with its apparent absence in the human, suggests its involvement in innate immunity. PMID:12370446

Non-random gamete fusion is one of several potential cryptic female choice mechanisms that have been postulated and that may enhance the survival probability of the offspring. Previous studies have found that gamete fusion in mice is influenced by genes of the major histocompatibility complex (MHC) region. Here we test (i) whether there is MHC-dependent gamete fusion in whitefish (Coregonus sp.) and (ii) whether there is a link between the MHC and embryo susceptibility to an infection by the bacterium Pseudomonas fuorescens. We experimentally bred whitefish and reared sibships in several batches that either experienced or did not experience strong selection by P. fluorescens. We then determined the MHC class II B1 genotype of 1016 surviving larvae of several full sibships. We found no evidence for MHC-linked gamete fusion. However, in one of seven sibships we found a strong connection between the MHC class II genotype and embryo susceptibility to P. fluorescens. This connection was still significant after correcting for multiple testing. Hence, the MHC class II genotype can considerably influence embryo survival in whitefish, but gamete fusion seems to be random with respect to the MHC.

All mature B cells coexpress major histocompatibility complex (MHC) class II molecules, I-A and I-E, which are restriction elements required for antigen presentation to CD4+ T cells. However, the expression of class II during the early stages of B cell development has been unclear. We demonstrate here that there is a difference in the expression of class II during murine B cell development in the fetal liver and adult bone marrow (BM). These differences define two distinct B cell developmental pathways. The Fetal-type (FT) pathway is characterized by pre-B and immature IgM+ B cells generated in the fetal liver which initially lack all class IIexpression. In contrast, the Adult-type (AT) pathway is typified by B cells developing in the adult BM which express class II molecules from the pre-B cell stage. In vitro stromal cell cultures of sorted fetal liver and adult BM pro-B cells indicated that the difference in I-A expression during B cell development is intrinsic to the progenitors. In addition, we show that FT B cell development is not restricted to the fetal liver but occurs in the peritoneal cavities, spleens, liver, and BM of young mice up to at least 1 mo of age. The AT B cell development begins to emerge after birth but is, however, restricted to the BM environment. These findings indicate that there are two distinct B cell developmental pathways during ontogeny, each of which could contribute differentially to the immune repertoire and thus the functions of B cell subsets and lineages. PMID:7913950

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs. PMID:26544200

The major histocompatibility complex (MHC) class I-related molecule, MR1, is highly conserved in mammals and can present bacteria-derived vitamin B metabolites to mucosal-associated invariant T (MAIT) cells, possibly having important defense function in the microbial infection. MR1B is a splice variant of MR1 and possesses an intriguing domain structure with only two extracellular domains resembling some NKG2D ligand molecules. Thus far, cell surface expression of MR1B could not be analyzed with flow cytometry due to a lack of appropriate antibodies reactive with MR1B. Here we clarified the expression of MR1B recombinant protein on the cell surface of the transfected cells by flow cytometry analyses using the antiserum against MR1. Consistently, MR1B tagged with FLAG peptide at the N-terminus also could be detected with anti-FLAG monoclonal antibodies. Our result showed that MR1B can be recognized on the cell surface by macromolecules such as antibodies, indicating its potential of interaction with certain receptor(s). We discuss possibility of interaction of MR1B and/or the full-length MR1 with some receptor(s) other than αβ T cell receptor (TCR) of MAIT cells based on the highly conserved characteristic residues of the ligand-binding domains of MR1 and its MAIT cells αβTCR footprints.

Genes encoded by the major histocompatibility complex (MHC) are crucial for the recognition and presentation of antigens to the immune system. In contrast to their closest relatives, chimpanzees and humans, much less is known about variation in gorillas at these loci. This study explored the exon 2 variation of -DPB1, -DQB1, and -DRB genes in 46 gorillas from four populations while simultaneously evaluating the feasibility of using fecal samples for high-throughput MHC genotyping. By applying strict similarity- and frequency-based analysis, we found, despite our modest sample size, a total of 18 alleles that have not been described previously, thereby illustrating the potential for efficient and highly accurate MHC genotyping from non-invasive DNA samples. We emphasize the importance of controlling for multiple potential sources of error when applying this massively parallel short-read sequencing technology to PCR products generated from low concentration DNA extracts. We observed pronounced differences in MHC variation between species, subspecies and populations that are consistent with both the ancient and recent demographic histories experienced by gorillas.

The effect of brain-enriched gangliosides on constitutive and cytokine- inducible expression of major histocompatibility complex (MHC) class I and II genes in cultured astrocytes was studied. Before treatment with gangliosides, astrocytes expressed constitutive MHC class I but not class II molecules, however, the expression of both MHC class I and II cell surface molecules on astrocytes was induced to high levels by interferon gamma (IFN-gamma). Constitutive and IFN-gamma-inducible expression of MHC class I and II molecules was suppressed by treatment of astrocytes with exogenous bovine brain gangliosides in a dose- dependent manner. Constitutive and induced MHC class I and II mRNA levels were also suppressed by gangliosides, indicating control through transcriptional mechanisms. This was consistent with the ability of gangliosides to suppress the binding activity of transcription factors, especially NF-kappa B-like binding activity, important for the expression of both MHC class I and II genes. These studies may be important for understanding mechanisms of central nervous system (CNS)- specific regulation of major histocompatibility molecules in neuroectodermal cells and the role of gangliosides in regulating MHC- restricted antiviral and autoimmune responses within the CNS. PMID:8376939

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian.

Cells expressing CD4, CD8, major histocompatibility complex (MHC) Class II, and macrophage biomarkers in lungs of chickens were quantified by measuring total area of antigen expressed using imageJ, a software program developed at the National Institutes of Health and available at no cost. The procedures reported here were rapid, and reproducible. Total area of antigen expressed had positive correlation with manual counts of cells expressing CD4 and CD8 biomarkers after inoculation with serotype 1 Marek's disease virus (MDV) vaccines. Visual inspection and overlays prepared from outlines of cells counted by imageJ confirmed agreement between antigen expression and area measured. Total area measured was not dependent on time of image acquisition from randomly selected fields from the same slides. Total area values were not computer specific, but acquisition of the original images required standardization of microscope used and camera setup. All steps in the process from sample collection through sectioning, staining, and image acquisition must be standardized as much as possible. Chickens infected with a very virulent+ (vv(+)) isolate of MDV (648A) had increased CD4, CD8, MHC Class II, and macrophage biomarker expression compared to noninfected control chickens at 10 days post infection, but variable responses depending on the specific biomarker measured at 3 and 5 days post infection. The procedure described here is faster and more reproducible than manual counting in cases (CD4 and CD8) where the number of positive cells is low enough for manual counts. Manual counting is not possible with MHC Class II and macrophage antigens nor when CD4(+) cells are present in large numbers following proliferation to tumors, thus subjective systems are used for scoring in these conditions. Using imageJ as described eliminates the need for subjective and less reproducible methods for measuring expression of these antigens.

The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

The endosomal site(s) where MHC class II molecules become competent to bind antigenic peptide has not been completely characterized. We identified endocytic compartments through which newly synthesized MHC class II molecules move prior to their expression on the plasma membrane. The compartments co-sediment with lysosomes in the most dense regions of Percoll gradients. The appearance of proteolytic fragments of the invariant chain (I chain), namely leupeptin-induced proteins (LIPs) and class-II-associated invariant chain peptides (CLIP), in this region of the gradient suggests that the release of MHC class II molecules from I chain association occurs within these vesicles. The formation of SDS-stable alpha beta dimers indicated that MHC class II molecules contained within these compartments are receptive to peptide binding. A majority of the HLA-DM protein was found in the same region of the Percoll gradient, consistent with its established function in MHC class-II-restricted antigen presentation. Immunoelectron micrographs of dense-sedimenting compartments indicated that I chain, MHC class II, and DM molecules are contained within both multivesicular and multilamellar vesicles. The final stages of I chain dissociation from MHC class II molecules and DM-mediated peptide loading probably occur in these compartments.

The use of major histocompatibility complex (MHC) tetramers in the detection and analysis of antigen-specific T cells has become more widespread since its introduction 11 years ago. Early challenges in the application of tetramer staining to CD4+ T cells centred around difficulties in the expression of various class IIMHC allelic variants and the detection of low-frequency T cells in mixed populations. As many of the technical obstacles to class IIMHC tetramer staining have been overcome, the focus has returned to uncertainties concerning how oligomer valency and T-cell receptor/MHC affinity affect tetramer binding. Such issues have become more important with an increase in the number of studies relying on direct ex vivo analysis of antigen-specific CD4+ T cells. In this review we discuss which problems in class IIMHC tetramer staining have been solved to date, and which matters remain to be considered. PMID:18251991

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.

Of the various molecular regulatory mechanisms that may be used by human trophoblast cells to down-regulate expression of HLA class I genes, we chose to investigate the methylation of DNA, generally associated with inhibition of transcription. We analyzed the methylation status of different HLA class I loci in villous and extravillous cytotrophoblast cells and in vitro-differentiated syncytiotrophoblast, purified from human term placenta, as well as in the human trophoblast-derived JAR and JEG-3 cell lines. We then compared methylation status and transcriptional activity. An inverse relationship was established between JAR and JEG-3: HLA-A, -B, and -G are methylated and repressed in JAR, whereas in JEG-3, HLA-A is methylated and repressed but HLA-B and -G are partially methylated and transcribed. HLA-E is unmethylated and transcribed in both cell lines. Apart from HLA-E, which is always unmethylated and transcribed, no such relationship exists for the other class I loci in trophoblast cells. Whereas nonclassical HLA-G and classical HLA-A and -B class I genes are undermethylated in both cytotrophoblast and syncytiotrophoblast, they are clearly transcribed in the former but minimally transcribed in the latter subpopulation. Thus, the down-regulation of class I gene expression in the in vitro-differentiated synctiotrophoblast is unlikely to be caused by DNA methylation. Furthermore, there is no detectable expression of any class I molecule at the cell surface of either trophoblast cell subpopulation, suggesting a negative control on translation and/or on the secretory pathway to the plasma membrane. 50 refs., 11 figs., 1 tab.

Major histocompatibility complex class I chain-related B (MICB) is a membrane-bound glycoprotein involved in both innate and adaptive immunity through its interaction with NKG2D receptors present on γδ T, αβ CD8(+) T, and natural killer cells. Factors known to upregulate MICB expression include heat shock, viral or bacterial infection, and tumorigenesis, and here, we explored the effect of 17β-estradiol (E2) on MICB regulation. Physiological concentrations of E2 were found to suppress MICB mRNA and surface protein levels and this effect was antagonized by the antiestrogen ICI 182780. The inhibitory effect of E2 was also observed for other NKG2D ligands, MICA and ULBPs. Evaluation of promoter fragments from the common MICB*00502 allele revealed that inhibition of transcription by E2 required the GC box at -87. The electrophoretic mobility shift assay and supershift analysis established the presence of SP1, SP3, or estrogen receptor α recognition sites within the MICB promoter sequence and interaction of these factors in situ was confirmed by chromatin immunoprecipitation. We conclude that E2 upon forming a complex with its cognate receptor suppresses MICB expression through binding with SP1/SP3 sites within the MICB promoter GC box. These results suggest that the partial benefit of 17β-estradiol on autoimmune diseases may be mediated by reducing the immune NKG2D ligands like MICB.

Three sequence lineages of MHC class I genes have been described in zebrafish (Danio rerio): U, Z, and L. The U lineage genes encoded on zebrafish chromosome 19 are predicted to provide the classical function of antigen presentation. This MHC class I locus displays significant haplotypic variation and is the only MHC class I locus in zebrafish that shares conserved synteny with the core mammalian MHC. Here we describe two MHC class I U lineage genes, mhc1ula and mhc1uma, that map to chromosome 22. Unlike the U lineage proteins encoded on chromosome 19, Ula and Uma likely play a nonclassical role as they lack conservation of key peptide binding residues, display limited polymorphic variation, and exhibit tissue-specific expression. We also describe a null haplotype at this chromosome 22 locus in which the mhc1ula and mhc1uma genes are absent due to a ∼30 kb deletion with no other MHC class I sequences present. Functional and non-functional transcripts of mhc1ula and mhc1uma were identified; however, mhc1uma transcripts were often not amplified or amplified at low levels from individuals possessing an apparently bona fide gene. These distinct U lineage genes may be restricted to the superorder Ostariophysi as similar sequences only could be identified from the blind cavefish (Astyanyx mexicanus), fathead minnow (Pimephales promelas), goldfish (Carassius auratus), and grass carp (Ctenopharyngodon idellus). PMID:26254596

The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation. PMID:27760184

By cross-hybridization in low stringency conditions, using a probe derived from an HLA-DQ beta cDNA clone, we have isolated several chicken genomic DNA clones. These clones were mapped to the major histocompatibility complex (MHC) of the chick (B complex) by virtue of their ability to detect restriction enzyme length polymorphisms between congenic lines of chicken. Evidence was obtained for the presence of at least three B-L beta genes in the chicken genome. The B-L beta genes are transcribed specifically in tissues containing cells of the B lymphocyte and myeloid lineages and expressing the B-L antigens. Exons encoding the beta 1, beta 2 and transmembrane domains of a B-L beta chain have been identified with 63, 66 and 62% similarity with the HLA-DQ beta sequence. This first isolation of an MHC class II gene outside of the mammalian class provides insight into the evolution of MHC genes based on the comparison of avian and mammalian class II beta chain amino acid and nucleotide sequences. Images PMID:2841107

We have known since the late 1980s that the function of classical major histocompatibility complex (MHC) class I molecules is to bind peptides and display them at the cell surface to cytotoxic T cells. Recognition by these sentinels of the immune system can lead to the destruction of the presenting cell, thus protecting the host from pathogens and cancer. Classical MHC class I molecules (MHC I hereafter) are co-dominantly expressed, polygenic, and exceptionally polymorphic and have significant sequence diversity. Thus, in most species, there are many different MHC I allotypes expressed, each with different peptide-binding specificity, which can have a dramatic effect on disease outcome. Although MHC allotypes vary in their primary sequence, they share common tertiary and quaternary structures. Here, we review the evidence that, despite this commonality, polymorphic amino acid differences between allotypes alter the ability of MHC I molecules to change shape (that is, their conformational plasticity). We discuss how the peptide loading co-factor tapasin might modify this plasticity to augment peptide loading. Lastly, we consider recent findings concerning the functions of the non-classical MHC I molecule HLA-E as well as the tapasin-related protein TAPBPR (transporter associated with antigen presentation binding protein-related), which has been shown to act as a second quality-control stage in MHC I antigen presentation. PMID:28299193

The major histocompatibility complex (MHC) is the center of the immune universe. Genes in the MHC determine which antigens are processed and presented. Not surprisingly, the MHC contributes the major genetic component to important autoimmune diseases and will no doubt, although evidence is limited, contribute to resistance to infectious disorders. Vertebrates all seem to have MHC genes and it should be possible to determine, within the next few years, whether the clustering of antigen processing and presenting genes in this region is a conserved feature. One could imagine an evolutionary advantage to maintaining the MHC as a unit, either to coordinate expression of the genes in different tissues, or to coordinate T-cell selection during thymic ontogeny, since inheriting a linked set of polymorphic gene products may help to avoid conflicts during positive and negative selection. 153 refs., 9 figs., 3 tabs.

Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4+ T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4+ T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway. PMID:26195549

Background The sympathetic neurotransmitter Norepinephrine (NE) contributes to tumorigenesis and cancer progression. This study aims to investigate the role of NE in modulating the immune phenotype and allowing pancreatic carcinoma (PC) cells to escape the immune response. Methods Varied concentrations of NE and interferon-gamma (IFN-γ) were administrated to MIA PaCa-2 and BxPC-3 cell lines for 48 hours. Proliferation and invasion were then investigated using an MTT assay and a membrane invasion culture system respectively. MHC-I, B7-1, IDO and B7-H1 expression were measured using real-time quantitative RT-PCR, western blotting and immunocytochemistry. The synergistic and time-dependent effects of NE/IFN-γ were also investigated. Adrenergic antagonists were used to identify the relevant target receptor of NE. Results The results showed that NE had dose-dependent and time-dependent effects on cell biological processes as well as on the expression of MHC-I, B7-1, IDO and B7-H1. These effects occurred mainly via the β2-adrenergic receptor. Long-term NE treatment was able to antagonize some of the effects of IFN-γ (after 2 weeks of treatment), but NE and IFN-γ had significant synergistic stimulatory effects on IDO and B7-H1 expression. The residual effects on biological activities lasted for 2 weeks, while the immunophenotypic changes decreased at early time points after treatment. Conclusions NE plays important roles in modulating PC cell biological activities and affecting MHC-I, B7-1, IDO and B7-H1 expression in vitro, mainly via the β2-adrenergic receptor (β2-AR) in a time- and dose-dependent fashion. Only at extended treatment durations could NE affect PC cell progression and immune evasion. PMID:23029049

Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors. PMID:25058148

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abβ0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints. PMID:9636191

The allelic diversity of the DRB locus in major histocompatibility complex (MHC) genes was analyzed in the brown bear (Ursus arctos) from the Hokkaido Island of Japan, Siberia, and Kodiak of Alaska. Nineteen alleles of the DRB exon 2 were identified from a total of 38 individuals of U. arctos and were highly polymorphic. Comparisons of non-synonymous and synonymous substitutions in the antigen-binding sites of deduced amino acid sequences indicated evidence for balancing selection on the bear DRB locus. The phylogenetic analysis of the DRB alleles among three genera (Ursus, Tremarctos, and Ailuropoda) in the family Ursidae revealed that DRB allelic lineages were not separated according to species. This strongly shows trans-species persistence of DRB alleles within the Ursidae.

MHC classical class I and class II genes have been identified in representative species from all major jawed vertebrate taxa, the oldest group being the cartilaginous fish, whereas no class I/II genes of any type have been detected in animals from older taxa. Among ectothermic vertebrate classes, studies of MHC architecture have been done in cartilaginous fish (sharks), bony fish (several teleost species), and amphibians (the frog Xenopus). The Xenopus MHC contains class I, class II, and class III genes, demonstrating that all of these genes were linked in the ancestor of the tetrapods, but the gene order is not the same as that in mouse/man. Studies of polyploid Xenopus suggest that MHC genes can be differentially silenced when multiple copies are present; i.e. MHC 'subregions' can be silenced. Surprisingly, in all teleosts examined to date class I and class II genes are not linked. Likewise, class III genes like the complement genes factor B (Bf) and C4 are scattered throughout the genome of teleosts. However, the presumed classical class I genes are closely linked to the 'immune' proteasome genes, LMP2 and LMP7, and to the peptide-transporter genes (TAP), implying that a true 'class I region' exists in this group. A similar type of linkage group is found in chickens and perhaps Xenopus, and thus it may reveal the ancestral organization of class I-associated genes. In cartilaginous fish, classical and non-classical class I genes have been isolated from three shark species, and class II A and B chain genes from nurse sharks. Studies of MHC linkage in sharks are being carried out to provide further understanding of the putative primordial organization of MHC Segregation studies in one shark family point to linkage of classical class I and class II genes, suggesting that the non-linkage of these genes in teleosts is a derived characteristic.

Current influenza vaccines are effective but imperfect, failing to cover against emerging strains of virus and requiring seasonal administration to protect against new strains. A key step to improving influenza vaccines is to improve our understanding of vaccine-induced protection. While it is clear that antibodies play a protective role, vaccine-induced CD8+ T cells can improve protection. To further explore the role of CD8+ T cells, we used a DNA vaccine that encodes antigen dimerized to an immune cell targeting module. Immunizing CB6F1 mice with the DNA vaccine in a heterologous prime-boost regime with the seasonal protein vaccine improved the resolution of influenza disease compared with protein alone. This improved disease resolution was dependent on CD8+ T cells. However, DNA vaccine regimes that induced CD8+ T cells alone were not protective and did not boost the protection provided by protein. The MHC-targeting module used was an anti-I-Ed single chain antibody specific to the BALB/c strain of mice. To test the role of MHC targeting, we compared the response between BALB/c, C57BL/6 mice, and an F1 cross of the two strains (CB6F1). BALB/c mice were protected, C57BL/6 were not, and the F1 had an intermediate phenotype; showing that the targeting of antigen is important in the response. Based on these findings, and in agreement with other studies using different vaccines, we conclude that, in addition to antibody, inducing a protective CD8 response is important in future influenza vaccines. PMID:27602032

Reduced immune gene diversity is thought to negatively affect the capacity of organisms to adapt to pathogen challenges, which represent a major force in natural selection. Genes of the Major Histocompatibility Complex (MHC) are the most widely invoked adaptive loci in conservation biology, and have become the most popular genetic markers to investigate pathogen-host interactions in vertebrates. Although MHC genes are the most polymorphic genes described in the vertebrate genome, the extent to which MHC diversity determines the long-term persistence of populations is, unclear and often debated, as recent studies have documented the occurrence of natural populations thriving even after a depletion of MHC diversity caused by genetic drift. Here, we show that some phylogenetically related species belonging to the Falco genus (Aves: Falconidae) present a dramatically low MHC variability that has not precluded, nevertheless, the successful colonization of almost all existing regions and habitats worldwide. We found evidence for two remarkably different patterns of MHC variation within the genus. While kestrels show a high MHC variation according to the general theory, falcons exhibit an ancestrally low intra- and inter-specific MHC allelic diversity. We provide compelling evidence that this pattern is not caused by the degeneration of functional genes into pseudogenes, the inadvertent analyses of paralogous MHC genes, or the devastating action of genetic drift. Instead, our results strongly support the idea of an evolutionary transition driven and maintained by natural selection from primarily highly variable towards low polymorphic, but functional and expressed, MHC genes with species-specific pathogen-recognition capabilities.

Small, isolated populations may experience increased extinction risk due to reduced genetic variability at important functional genes, thus decreasing the population's adaptive potential. The major histocompatibility complex (MHC), a key immunological gene cluster, usually shows high variability maintained by positive or balancing selection in response to challenges by pathogens. Here we investigated for the first time, the variability of 3 MHC class II genes (DRB1, DQA1, and DQB1) in 94 samples collected from Italian wolves. The Italian wolf population has been long isolated south of the Alps and is presently recovering from a recent bottleneck that decreased the population to less than 100 individuals. Despite the bottleneck, Italian wolves show remarkable MHC variability with 6-9 alleles per locus, including 2 recently described alleles at DRB1. MHC sequences show signatures of historical selective pressures (high d N/d S ratio, ω > 1.74) but no evidence of ongoing selection. Variation at the MHC genes and 12 background microsatellite loci were not apparently affected by the recent bottleneck. Although MHC alleles of domestic dog origin were detected in 8 genetically admixed individuals, these alleles were rare or absent in nonadmixed wolves. Thus, despite known hybridization events between domestic dogs and Italian wolves, the Italian wolf population does not appear affected by deep introgression of domestic dog MHC alleles.

The survival of naïve T cells is compromised in the absence of molecules encoded by the major histocompatibility complex (MHC) while antigen-experienced T cells survive. We hypothesized that survival pressures in an in vivo, MHC-deficient environment would permit enrichment of less frequent antigen-experienced autoreactive cells at the expense of the majority of antigen naïve T cells. To test this hypothesis, we generated MHC class I and class II-deficient mice in NOD and C57Bl/6 (B6) backgrounds, and examined the capacity of adoptively transferred autoimmune-prone NOD T cells, or non-autoimmune prone naïve B6 T cells, respectively, to reject transplanted wild type pancreatic islets or transplantable tumors in the MHC-deficient mice. In the MHC-deficient environment, CD4 T cells acquired self-hostile properties (islet rejection and tumor invasion) that were independent from their genetic propensity for autoreactivity, while CD8 T cells required appropriate prior exposure to antigen in order to survive and function (reject tumor) in this environment; however, disengagement of Tob1, a negative regulator of proliferation, led to a reverse phenotype with regard to persistence of CD4 and CD8 T cells in the MHC-deficient environment. Our data suggest that self-peptide/MHC interactions have dual roles to facilitate survival and restrain autoreactivity, thus acting as integral components of an intrinsic network of negative regulation that maintains tolerance. PMID:18523772

An emerging molecular mechanism by which docosahexaenoic acid (DHA) exerts its effects is modification of lipid raft organization. The biophysical model, based on studies with liposomes, shows that DHA avoids lipid rafts because of steric incompatibility between DHA and cholesterol. The model predicts that DHA does not directly modify rafts; rather, it incorporates into nonrafts to modify the lateral organization and/or conformation of membrane proteins, such as the major histocompatibility complex (MHC) class I. Here, we tested predictions of the model at a cellular level by incorporating oleic acid, eicosapentaenoic acid (EPA), and DHA, compared with a bovine serum albumin (BSA) control, into the membranes of EL4 cells. Quantitative microscopy showed that DHA, but not EPA, treatment, relative to the BSA control diminished lipid raft clustering and increased their size. Approximately 30% of DHA was incorporated directly into rafts without changing the distribution of cholesterol between rafts and nonrafts. Quantification of fluorescence colocalization images showed that DHA selectively altered MHC class I lateral organization by increasing the fraction of the nonraft protein into rafts compared with BSA. Both DHA and EPA treatments increased antibody binding to MHC class I compared with BSA. Antibody titration showed that DHA and EPA did not change MHC I conformation but increased total surface levels relative to BSA. Taken together, our findings are not in agreement with the biophysical model. Therefore, we propose a model that reconciles contradictory viewpoints from biophysical and cellular studies to explain how DHA modifies lipid rafts on several length scales. Our study supports the notion that rafts are an important target of DHA's mode of action.

Females of several vertebrate species selectively mate with males on the basis of the major histocompatibility complex (MHC) genes. As androgen-mediated maternal effects have long-lasting consequences for the adult phenotype, both mating and reproductive success may depend on the combined effect of MHC genotype and exposure to androgens during early ontogeny. We studied how MHC-based mate choice in ring-necked pheasants (Phasianus colchicus) was influenced by an experimental in ovo testosterone (T) increase. There was no conclusive evidence of in ovo T treatment differentially affecting mate choice in relation to MHC genotype. However, females avoided mating with males with a wholly different MHC genotype compared with males sharing at least one MHC allele. Females also tended to avoid mating with MHC-identical males, though not significantly so. These findings suggest that female pheasants preferred males with intermediate MHC dissimilarity. Male MHC heterozygosity or diversity did not predict the expression of ornaments or male dominance rank. Thus, MHC-based mating preferences in the ring-necked pheasant do not seem to be mediated by ornaments' expression and may have evolved mainly to reduce the costs of high heterozygosity at MHC loci for the progeny, such as increased risk of autoimmune diseases or disruption of coadapted gene pools.

Shaping natural killer (NK) cell functions in human immunity and reproduction are diverse killer cell immunoglobulin-like receptors (KIRs) that recognize polymorphic MHC class I determinants. A survey of placental mammals suggests that KIRs serve as variable NK cell receptors only in certain primates and artiodactyls. Divergence of the functional and variable KIRs in primates and artiodactyls predates placental reproduction. Among artiodactyls, cattle but not pigs have diverse KIRs. Catarrhine (humans, apes, and Old World monkeys) and platyrrhine (New World monkeys) primates, but not prosimians, have diverse KIRs. Platyrrhine and catarrhine systems of KIR and MHC class I are highly diverged, but within the catarrhines, a stepwise co-evolution of MHC class I and KIR is discerned. In Old World monkeys, diversification focuses on MHC-A and MHC-B and their cognate lineage II KIR. With evolution of C1-bearing MHC-C from MHC-B, as informed by orangutan, the focus changes to MHC-C and its cognate lineage III KIR. Evolution of C2 from C1 and fixation of MHC-C drove further elaboration of MHC-C-specific KIR, as exemplified by chimpanzee. In humans, the evolutionary trajectory changes again. Emerging from reorganization of the KIR locus and selective attenuation of KIR avidity for MHC class I are the functionally distinctive KIR A and KIR B haplotypes.

Summary Shaping natural killer (NK) cell functions in human immunity and reproduction are diverse killer-cell immunoglobulin-like receptors (KIRs) that recognize polymorphic MHC class I determinants. A survey of placental mammals suggests KIRs serve as variable NK cell receptors only in certain primates and artiodactyls. Divergence of functional and variable KIRs in primates and artiodactyls predates placental reproduction. Among artiodactyls, cattle but not pigs have diverse KIRs. Catarrhine (humans, apes, and Old World monkeys) and platyrrhine (New World monkeys) primates, but not prosimians, have diverse KIRs. Platyrrhine and catarrhine systems of KIR and MHC class I are highly diverged, but within the catarrhines a stepwise co-evolution of MHC class I and KIRs is discerned. In Old World monkeys, diversification focuses on MHC-A and MHC-B and their cognate lineage II KIR. With evolution of C1-bearing MHC-C from MHC-B, as informed by orangutan, the focus changes to MHC-C and its cognate lineage III KIR. Evolution of C2 from C1 and fixation of MHC-C, drove further elaboration of MHC-C-specific KIRs, as exemplified by chimpanzee. In humans, the evolutionary trajectory changes again. Emerging from reorganization of the KIR locus and selective attenuation of KIR avidity for MHC class I are the functionally distinctive KIR A and KIR B haplotypes. PMID:26284483

Summary The structure and amino acid diversity of the T-cell receptor (TCR), similar in nature to that of Fab portions of antibodies, would suggest these proteins have a nearly infinite capacity to recognize antigen. Yet all currently defined native T cells expressing an α and β chain in their TCR can only sense antigen when presented in the context of a major histocompatibility complex (MHC) molecule. This MHC molecule can be one of many that exist in vertebrates, presenting small peptide fragments, lipid molecules, or small molecule metabolites. Here we review the pattern of TCR recognition of MHC molecules throughout a broad sampling of species and T-cell lineages and also touch upon T cells that do not appear to require MHC presentation for their surveillance function. We review the diversity of MHC molecules and information on the corresponding T-cell lineages identified in divergent species. We also discuss TCRs with structural domains unlike that of conventional TCRs of mouse and human. By presenting this broad view of TCR sequence, structure, domain organization, and function, we seek to explore how this receptor has evolved across time and been selected for alternative antigen-recognition capabilities in divergent lineages. PMID:26284470

HGP-30, a 30 amino acid synthetic peptide homologous to a conserved region of HIV-1(SF2) p17 (aa86-115), has previously been shown to elicit both cellular and humoral immune responses when conjugated to KLH and adsorbed to alum. However, the free HGP-30 peptide is not immunogenic in animals. In order to improve the immunogenicity of HGP-30, peptide conjugates consisting of a modified HGP-30 sequence (m-HGP-30/aa82-111) and a peptide segment, residues 38-50, of the MHC I accessory molecule, human beta-2-microglobulin (beta-2-M), referred to as Peptide J, or a peptide from the MHCII beta chain (peptide G) were evaluated in mice. The effects of carriers and adjuvants on serum antibody titers, specificities to various HIV-1 clade peptides similar to HGP-30 and isotype patterns were examined. Peptides J or especially G conjugated to modified-HGP-30 (LEAPS 102 and LEAPS 101, respectively) generated comparable or better immune responses to modified HGP-30 than KLH conjugates as judged by the induction of: (1) similar antibody titers; (2) broader HIV clade antigen binding; and (3) antibody isotype response patterns indicative of a TH1 pathway (i.e. increased amounts of IgG2a and IgG2b antibodies). The ISA 51 and MPL(R)-SE adjuvants induced higher antibody responses than alum, with the ISA 51 being more potent. Immune responses to LEAPS 102, as compared to LEAPS 101, were weaker and slower to develop as determined by antibody titers and cross clade reactivity of the antibodies induced. Compared to KLH conjugates which induced significant anti-KLH antibody titers, minimal antibody responses were observed to peptide G, the more immunogenic conjugate, and peptide J. These results suggest that modified HGP-30 L.E.A.P.S. constructs may be useful as HIV vaccine candidates for preferential induction of TH1 directed cell mediated immune responses.

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage− were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 ± 0.06% of the PB nucleated cells and 55.9 ± 11.9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 ± 0.04% of PB nucleated cells and 44.53 ± 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor α-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1β (97 ± 5% of positive cells), IL-6 (96 ± 1.1% of positive cells), IL-12 (81.5 ± 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-α) (84 ± 22.1% of positive cells) as well as chemokines such as IL-8 (99 ± 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8+ T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing. PMID:26869717

Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8+ T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Autoimmune diseases (ADs), or autoinflammatoiy diseases, are growing in complexity as diagnoses improve and many factors escalate disease risk. Considerable genetic similarity is found among ADs, and they are frequently associated with major histocompatibility complex (MHC) genes. However, a given disease may be associated with more than one human leukocyte antigen (HLA) allotype, and a given HLA may be associated with more than one AD. The associations of non-MHC genes with AD present an additional problem, and the situation is further complicated by the role that other factors, such as age, diet, therapeutic drugs, and regional influences, play in disease. This review discusses some of the genetics and biochemistry of HLA-linked AD and inflammation, covering some of the best-studied examples and summarizing indicators for class I- and II-mediated disease. However, the scope of this review limits a detailed discussion of all known ADs.

Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.

The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHCII) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHCII in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHCII-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHCII. Whereas binding was undetectable in solution, the affinity of the CD4/pMHCII interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/μm2. This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHCII binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2–20% increase in the effective affinity of the TCR for pMHCII. The affinity of CD4/pMHCII therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination. PMID:27114505

Marek׳s disease virus (MDV) is a widespread α-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198-205 (2001)), but the gene(s) involved have not been identified. Here we demonstrate that an MDV gene, MDV012, is capable of reducing surface expression of MHC class I on chicken cells. Co-expression of an MHC class I-binding peptide targeted to the endoplasmic reticulum (bypassing the requirement for the TAP peptide transporter) partially rescued MHC class I expression in the presence of MDV012, suggesting that MDV012 is a TAP-blocking MHC class I immune evasion protein. This is the first unique non-mammalian MHC class I immune evasion gene identified, and suggests that α-herpesviruses have conserved this function for at least 100 million years.

Human T-lymphotropic virus type-1 (HTLV-1) is a complex retrovirus that causes adult T-cell leukemia/lymphoma (ATL) and is implicated in a variety of lymphocyte-mediated disorders. HTLV-1 pX ORF II encodes two proteins, p13II and p30II whose roles are beginning to be defined in the virus life cycle. Previous studies indicate the importance of these viral proteins in the ability of the virus to maintain viral loads and persist in an animal model of HTLV-1 infection. Intriguing new studies indicate that p30II is a multifunctional regulator that differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein (CBP)/p300 and specifically binds and represses tax/rex mRNA nuclear export. A new study characterized the role of p30II in regulation of cellular gene expression using comprehensive human gene arrays. Interestingly, p30II is an overall repressor of cellular gene expression, while selectively favoring the expression of regulatory gene pathways important to T lymphocytes. These new findings suggest that HTLV-1, which is associated with lymphoproliferative diseases, uses p30II to selectively repress cellular and viral gene expression to favor the survival of cellular targets ultimately resulting in leukemogenesis.

Recent computational approaches in bioinformatics can achieve high performance, by which they can be a powerful support for performing real biological experiments, making biologists pay more attention to bioinformatics than before. In immunology, predicting peptides which can bind to MHC alleles is an important task, being tackled by many computational approaches. However, this situation causes a serious problem for immunologists to select the appropriate method to be used in bioinformatics. To overcome this problem, we develop an ensemble prediction-based Web server, which we call MetaMHCpan, consisting of two parts: MetaMHCIpan and MetaMHCIIpan, for predicting peptides which can bind MHC-I and MHC-II, respectively. MetaMHCIpan and MetaMHCIIpan use two (MHC2SKpan and LApan) and four (TEPITOPEpan, MHC2SKpan, LApan, and MHC2MIL) existing predictors, respectively. MetaMHCpan is available at http://datamining-iip.fudan.edu.cn/MetaMHCpan/index.php/pages/view/info .

The BioBreeding/Worcester (BB/Wor) rat develops a spontaneous disorder that closely resembles human insulin-dependent (Type I) diabetes mellitus. The syndrome is preceded by lymphocytic insulitis that destroys pancreatic beta cells. The morphologic features of the spontaneous insulitis lesions are also observed within islets transplanted beneath the renal capsule of diabetes-prone and diabetic animals. This study reports the results of experiments in which immunohistochemical techniques were used to characterize the phenotype of the infiltrating mononuclear cells and detect the expression of class I and class IIMHC antigens in native islets and islet transplants in diabetic and diabetes-prone BB/Wor rats. The infiltrates within native pancreatic islets and islet grafts were comprised predominantly of Ia+ cells (dendritic cells and macrophages) CD4+ cells (helper/inducer lymphocytes and macrophages), CD5+ (pan-T) cells and smaller numbers of CD8+ (cytotoxic/suppressor and NK) cells. Pancreatic and graft insulitis were accompanied by markedly enhanced class I antigen expression on islet and exocrine cells. Class II (Ia) antigens were not detected on normal islet cells, islets undergoing insulitis or on islet transplants subjected to immune attack. In islet grafts stained with polymorphic MAbs that distinguish Ia antigens of donor and host origin, Ia antigen expression was limited to infiltrating dendritic cells and macrophages of host origin. It is concluded that the phenotypes of infiltrating mononuclear cells that comprise the insulitis lesion in spontaneous BB/Wor diabetes, and the inflammatory attack on islets transplanted into diabetic BB/Wor rats are the same, that pancreatic islet and graft insulitis occur in the presence of enhanced class I antigen expression but in the absence of class II antigen expression, and that infiltrating Ia+ cells within islet grafts are exclusively of recipient (BB/Wor) origin and may explain the initiation of immune insulitis

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as “NLRC5” [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8+ cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers. PMID:27162338

Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.

Antigen processing for presentation by major histocompatibility complex class II (MHCII) molecules requires the latter to travel through the endocytic pathway together with its chaperons: the invariant chain (Ii) and DM. Nevertheless, the nature of the compartments where MHCII molecules travel to acquire peptides lacks definition regarding molecules involved in intracellular vesicular trafficking, such as Rab small GTPases. We aimed to define which Rab proteins are present during the intracellular transport of MHCII, DM, and Ii through the endocytic pathway on their route to the cell surface during dendritic cell (DC) maturation. We examined, by means of three-color confocal microscopy, the association of MHCII, DM, and Ii with Rab5, Rab7, Rab9, and Rab11 during the maturation of bone marrow-derived or spleen DC in response to LPS as an inflammatory stimulus. Prior to the stage of immature DC, MHCII migrated from diffuse small cytoplasmic vesicles, predominantly Rab5+Rab7− and Rab5+Rab7+ into a pericentriolar Rab5+Rab7+Rab9+ cluster, with Rab11+ areas. As DC reached the mature phenotype, MHCII left the pericentriolar endocytic compartments toward the cell surface in Rab11+ and Rab9+Rab11+ vesicles. The invariant chain and MHCII transport pathways were not identical. DM and MHCII appeared to arrive to pericentriolar endocytic compartments of immature DC through partially different routes. The association of MHCII molecules with distinct Rab GTPases during DC maturation suggests that after leaving the biosynthetic pathway, MHCII sequentially traffic from typical early endosomes to multivesicular late endosomes to finally arrive at the cell surface in Rab11+ recycling-type endosomes. In immature DCs, DM encounters transiently MHCII in the Rab5+Rab7+Rab9+ compartments, to remain there in mature DC.

Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.

Carnitine palmitoyltransferase (CPT) II deficiency, an inherited disorder of mitochondrial long-chain fatty-acid (LCFA) oxidation, results in two distinct clinical act phenotypes, namely, an adult (muscular) form and an infantile (hepatocardiomuscular) form. The rationale of this phenotypic heterogeneity is poorly understood. The adult form of the disease is commonly ascribed to the Ser-113-Leu substitution in CPT II. Only few data are available regarding the molecular basis of the infantile form of the disease. We report herein a homozygous A-2399-C transversion predicting a Tyr-628-Ser substitution in a CPT II-deficient infant. In vitro expression of mutant cDNA in COS-1 cells demonstrated the responsibility of this mutation for the disease. Metabolic consequences of the Ser-113-Leu and Tyr-628-Ser substitutions were studied in fibroblasts. The Tyr-628-Ser substitution (infantile form) resulted in a 10% CPT II residual activity, markedly impairing LCFA oxidation, whereas the Ser-113-Leu substitution (adult form) resulted in a 20% CPT II residual activity, without consequence on LCFA oxidation. These data show that CPT II activity has to be reduced below a critical threshold in order for LCFA oxidation in fibroblasts to be impaired. The hypothesis that this critical threshold differs among tissues could provide a basis to explain phenotypic heterogeneity of CPT II deficiency. 32 refs., 5 figs.

T-cell receptor (TCR) with unique major histocompatibility complex (MHC)-unrestricted antigen-binding properties was isolated from a human T-cell clone specific for the tumor antigen MUC1. This TCR binds its epitope on the MUC1 protein without the requirement of processing and presentation. A single-chain Vα/Vβ/Cβ (scTCR) was fused to a CD3 zeta (ζ) chain to allow expression on the surface of cells of the innate (granulocytes, macrophages, natural killer [NK] cells) as well as the adaptive (T and B cells) immune system. To test the ability of the cells of the innate immune system to reject a tumor when provided with a tumor antigen-specific TCR, we reconstituted severe combined immunodeficiency (SCID) mice with bone marrow cells transduced with a retroviral vector encoding this receptor and challenged them with a MUC1-positive human tumor. These mice controlled the growth of the tumor significantly better than the control mice. We performed a similar experiment in immunocompetent mice transgenic for human MUC1. Expression of the TCR on large percentages of cells did not result in infiltration or destruction of tissues expressing MUC1. Reconstituted mice controlled the outgrowth of a MUC1-transfected but not the parental control tumor. scTCR expression appears lifelong, suggesting a successful transduction of the self-renewing stem cells. (Blood. 2005;105:4583-4589) PMID:15746083

Summary Induced pluripotent stem cells (iPSCs) can serve as a source of cardiomyocytes (CMs) to treat end-stage heart failure; however, transplantation of genetically dissimilar iPSCs even within species (allogeneic) can induce immune rejection. We hypothesized that this might be limited by matching the major histocompatibility complex (MHC) antigens between the donor and the recipient. We therefore transplanted fluorescence-labeled (GFP) iPSC-CMs donated from a macaque with homozygous MHC haplotypes into the subcutaneous tissue and hearts of macaques having heterozygous MHC haplotypes (MHC-matched; group I) or without identical MHC alleles (group II) in conjunction with immune suppression. Group I displayed a higher GFP intensity and less immune-cell infiltration in the graft than group II. However, MHC-matched transplantation with single or no immune-suppressive drugs still induced a substantial host immune response to the graft. Thus, the immunogenicity of allogeneic iPSC-CMs was reduced by MHC-matched transplantation although a requirement for appropriate immune suppression was retained for successful engraftment. PMID:26905198

Although the existence of an immune response against tumor cells is well documented, the fact that tumors take off in cancer patients indicates that neoplastic cells can circumvent this response. Over the years many investigators have described strategies to rescue the anti-tumor immune response with the aim of creating specific and long-lasting protection against the disease. When exported to human clinical settings, these strategies have revealed in most cases a very limited, if any, positive outcome. We believe that the failure is mostly due to the inadequate triggering of the CD4+ T helper (TH) cell arm of the adaptive immunity, as TH cells are necessary to trigger all the immune effector mechanisms required to eliminate tumor cells. In this review, we focus on novel strategies that by stimulating MHC class II-restricted activation of TH cells generate a specific and persistent adaptive immunity against the tumor. This point is of critical importance for both preventive and therapeutic anti-tumor vaccination protocols, because adaptive immunity with its capacity to produce specific, long-lasting protection and memory responses is indeed the final goal of vaccination. We will discuss data from our as well as other laboratories which strongly suggest that triggering a specific and persistent anti-tumor CD4+ TH cell response stably modify not only the tumor microenvironment but also tumor-dependent extratumor microenvironments by eliminating and/or reducing the blood-derived tumor infiltrating cells that may have a pro-tumor growth function such as regulatory CD4+/CD25+ T cells and myeloid-derived-suppressor cells. Within this frame, therefore, we believe that the establishment of a pro-tumor environment is not the cause but simply the consequence of the tumor strategy to primarily counteract components of the adaptive cellular immunity, particularly TH lymphocytes. PMID:24600588

Tegumentary leishmaniasis (TL) constitutes a major public health problem with significant morbidity worldwide. Synthetic peptide-based vaccines are attractive candidates to protect against leishmaniasis, since T cell-specific epitopes can be delivery to antigen-presenting cells, leading to the generation of a Th1 cell-mediated immunity. In this context, the present study aims to evaluate the immunogenicity and protective efficacy of a vaccine composed of major histocompatibility complex class I and II-restricted epitopes derived from four Leishmania infantum proteins to protect mice against Leishmania amazonensis infection. This recombinant fusion protein was administered in BALB/c mice alone or with saponin. As controls, animals received saline or saponin. In the results, the administration of the recombinant protein plus saponin induced a specific IFN-γ, IL-12 and GM-CSF production, as well as high IgG2a isotype antibody levels, which protected mice against a challenge using L. amazonensis promastigotes. Lower parasite burden was found in the infected footpads, liver, spleen and draining lymph node of vaccinated mice, when compared to those from the control groups. In addition, protection was associated with a lower IL-4 and IL-10 response, which was accompanied by the antileishmanial nitrite production by spleen cells of the animals. Interestingly, the recombinant protein administered alone induced a partial protection against challenge. In conclusion, this study shows a new vaccine candidate based on T cell-specific epitopes that was able to induce protection against L. amazonensis infection.

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. In this study, we sequenced alleles of the BoLA class II loci, BoLA-DRB3 and BoLA-DQA1, from 650 Japanese cattle from six herds [three herds (507 animals) of Japanese Black cattle and three herds (143 animals) of Holstein cattle] using polymerase chain reaction-sequence-based typing (PCR-SBT) methods. We identified 26 previously reported distinct DRB3 alleles in the two populations: 22 in Japanese Black and 17 in Holstein. The number of DRB3 alleles detected in each herd ranged from 9 to 20. Next, we identified 15 previously reported distinct DQA1 alleles: 13 in Japanese Black and 10 in Holstein. The number of alleles in each herd ranged from 6 to 10. Thus, allelic divergence is significantly greater for DRB3 than for DQA1. A population tree on the basis of the frequencies of the DRB3 and DQA1 alleles showed that, although the genetic distance differed significantly between the two cattle breeds, it was closely related within the three herds of each breed. In addition, Wu-Kabat variability analysis indicated that the DRB3 gene was more polymorphic than the DQA1 gene in both breeds and in all herds, and that the majority of the hypervariable positions within both loci corresponded to pocket-forming residues. The DRB3 and DQA1 heterozygosity for both breeds within each herd were calculated based on the Hardy-Weinberg equilibrium. Only one Japanese Black herd showed a significant difference between the expected and observed heterozygosity at both loci. This is the first report presenting a detailed study of the allelic distribution of BoLA-DRB3 and -DQA1 genes in Japanese Black and Holstein cattle from different farms in Japan. These results may help to develop improved livestock breeding strategies in the future.

Antigenic peptides presented by MHC class I molecules are generated mainly by the proteasome in the cytosol. Several cytosolic aminopeptidases further trim proteasomal products to form mature epitopes or individual amino acids. However, the distinct function of cytosolic aminopeptidases in MHC class I Ag processing remains to be elucidated. In this study, we show that cytosolic aminopeptidases differentially affect the cell surface expression of MHC class I molecules in an allele-dependent manner in human cells. In HeLa cells, knockdown of puromycin-sensitive aminopeptidase (PSA) by RNA interference inhibited optimal peptide loading of MHC class I molecules, and their cell surface expression was correspondingly reduced. In contrast, depletion of bleomycin hydrolase (BH) enhanced optimal peptide loading and cell surface expression of MHC class I molecules. We did not find evidence on the effect of leucine aminopeptidase knockdown on the MHC class I Ag presentation. Moreover, we demonstrated that PSA and BH influence the peptide loading and surface expression of MHC class I in an allele-specific manner. In the absence of either PSA or BH, the surface expression and peptide-dependent stability of HLA-A68 were reduced, whereas those of HLA-B15 were enhanced. The surface expression and peptide-dependent stability of HLA-A3 were enhanced by BH knockdown, although those of HLA-B8 were increased in PSA-depleted conditions.

highlights the common mechanistic principles shared by the MHC I editors Tsn and TAPBPR, and the MHCII editor HLA-DM, but also illustrate the distinct quality control strategies employed by these chaperones to sample epitopes. Unraveling the mechanistic underpinnings of catalyzed peptide proofreading will be crucial for a thorough understanding of many aspects of immune recognition, from infection control and tumor immunity to autoimmune diseases and transplant rejection.

highlights the common mechanistic principles shared by the MHC I editors Tsn and TAPBPR, and the MHCII editor HLA-DM, but also illustrate the distinct quality control strategies employed by these chaperones to sample epitopes. Unraveling the mechanistic underpinnings of catalyzed peptide proofreading will be crucial for a thorough understanding of many aspects of immune recognition, from infection control and tumor immunity to autoimmune diseases and transplant rejection. PMID:28228754

experimental period and developed terminal disease. Our results indicate that the differences in immunoresponse were due to differences in major histocompatibility complex class II alleles and reflected the risk of BLV-induced leukemogenesis. In addition, it appears that susceptibility to tumor development may be determined to some extent by polymorphic residues binding to antigenic peptides directly within the binding cleft of the OLA-DR molecule.

Intratumor heterogeneity among cancer cells is promoted by reversible or irreversible genetic alterations and by different microenvironmental factors. There is considerable experimental evidence of the presence of a variety of malignant cell clones with a wide diversity of major histocompatibility class I (MHC-I) expression during early stages of tumor development. This variety of MHC-I phenotypes may define the evolution of a particular tumor. Loss of MHC-I molecules frequently results in immune escape of MHC-negative or -deficient tumor cells from the host T cell-mediated immune response. We review here the results obtained by our group and other researchers in animal models and humans, showing how MHC-I intratumor heterogeneity may affect local oncogenicity and metastatic progression. In particular, we summarize the data obtained in an experimental mouse cancer model of a methylcholanthrene-induced fibrosarcoma (GR9), in which isolated clones with different MHC-I expression patterns demonstrated distinct local tumor growth rates and metastatic capacities. The observed "explosion of diversity" of MHC-I phenotypes in primary tumor clones and the molecular mechanism ("hard"/irreversible or "soft"/reversible) responsible for a given MHC-I alteration might determine not only the metastatic capacity of the cells but also their response to immunotherapy. We also illustrate the generation of further MHC heterogeneity during metastatic colonization and discuss different strategies to favor tumor rejection by counteracting MHC-I loss. Finally, we highlight the role of MHC-I genes in tumor dormancy and cell cycle control.

T cell-mediated recognition of peptide-major histocompatibility complex (pMHC) class I and II molecules is crucial for the control of intracellular pathogens and cancer, as well as for stimulation and maintenance of efficient cytotoxic responses. Such interactions may also play a role in the development of autoimmune diseases. Novel insights into this mechanism are crucial to understanding disease development and establishing new treatment strategies. MHC multimers have been used for detection of antigen-responsive T cells since the first report by Altman et al. showed that tetramerization of pMHC class I molecules provided sufficient stability to T cell receptor (TCR)-pMHC interactions, allowing detection of MHC multimer-binding T cells using flow cytometry. Since this breakthrough the scientific community has aimed for expanding the capacity of MHC multimer-based detection technologies to facilitate large-scale epitope discovery and immune monitoring in limited biological material. Screening of T cell specificity using large libraries of pMHC molecules is suitable for analyses of T cell recognition potentially at genome-wide levels rather than analyses restricted to a selection of model antigens. Such strategies provide novel insights into the immune specificities involved in disease development and response to immunotherapy, and extend fundamental knowledge related to T cell recognition patterns and cross-recognition by TCRs. MHC multimer-based technologies have now evolved from detection of 1-2 different T cell specificities per cell sample, to include more than 1000 evaluable pMHC molecules using novel technologies. Here, we provide an overview of MHC multimer-based detection technologies developed over two decades, focusing primarily on MHC class I interactions.

Fowl adenoviruses (FAdVs) are the ethiologic agents of multiple pathologies in chicken. There are five different species of FAdVs grouped as FAdV-A, FAdV-B, FAdV-C, FAdV-D, and FAdV-E. It is of interest to develop immunodiagnostics and vaccine candidate for Peruvian FAdV-C in chicken infection using MHC restricted short peptide candidates. We sequenced the complete genome of one FAdV strain isolated from a chicken of a local farm. A total of 44 protein coding genes were identified in each genome. We sequenced twelve Cobb chicken MHC alleles from animals of different farms in the central coast of Peru, and subsequently determined three optimal human MHC-I and four optimal human MHC-II substitute alleles for MHC-peptide prediction. The potential MHC restricted short peptide epitope-like candidates were predicted using human specific (with determined suitable chicken substitutes) NetMHCMHC-peptide prediction model with web server features from all the FAdV genomes available. FAdV specific peptides with calculated binding values to known substituted chicken MHC-I and MHC-II were further filtered for diagnostics and potential vaccine epitopes. Promiscuity to the 3/4 optimal human MHC-I/II alleles and conservation among the available FAdV genomes was considered in this analysis. The localization on the surface of the protein was considered for class II predicted peptides. Thus, a set of class I and class II specific peptides from FAdV were reported in this study. Hence, a multiepitopic protein was built with these peptides, and subsequently tested to confirm the production of specific antibodies in chicken.

Fowl adenoviruses (FAdVs) are the ethiologic agents of multiple pathologies in chicken. There are five different species of FAdVs grouped as FAdV-A, FAdV-B, FAdV-C, FAdV-D, and FAdV-E. It is of interest to develop immunodiagnostics and vaccine candidate for Peruvian FAdV-C in chicken infection using MHC restricted short peptide candidates. We sequenced the complete genome of one FAdV strain isolated from a chicken of a local farm. A total of 44 protein coding genes were identified in each genome. We sequenced twelve Cobb chicken MHC alleles from animals of different farms in the central coast of Peru, and subsequently determined three optimal human MHC-I and four optimal human MHC-II substitute alleles for MHC-peptide prediction. The potential MHC restricted short peptide epitope-like candidates were predicted using human specific (with determined suitable chicken substitutes) NetMHCMHC-peptide prediction model with web server features from all the FAdV genomes available. FAdV specific peptides with calculated binding values to known substituted chicken MHC-I and MHC-II were further filtered for diagnostics and potential vaccine epitopes. Promiscuity to the 3/4 optimal human MHC-I/II alleles and conservation among the available FAdV genomes was considered in this analysis. The localization on the surface of the protein was considered for class II predicted peptides. Thus, a set of class I and class II specific peptides from FAdV were reported in this study. Hence, a multiepitopic protein was built with these peptides, and subsequently tested to confirm the production of specific antibodies in chicken. PMID:26664030

The restriction specificities of maturing thymocytes are determined by the Class IIMHC antigens expressed by non-lymphoid thymic tissues. The proliferative response of mature T lymphocytes to antigen-presenting cells (APC) and antigen requires that the APC express the same MHC antigens as the thymus in which the T cells differentiated. Thus, in the two-way bone marrow chimera (A + B----(A x B)F1), T lymphocyte populations of A and B haplotypes have each acquired the potential to recognize antigens associated with either parental haplotype. In spite of the large body of work on MHC restriction, we still do not have a clear understanding of the mechanisms which impose self restriction. The chimeric model systems used previously to study MHC restriction have used adult bone marrow cells as the source of lymphoid precursors. During normal ontogeny, T cells are derived from precursors in the fetal liver and we felt that a direct comparison of T cells from fetal liver and bone marrow-repopulated animals would shed light on the development of MHC restriction specificities during T cell ontogeny in the thymus or prethymically. We found that parental T lymphocyte populations isolated from two-way fetal liver chimeras cooperated only with syngeneic APC, while those from bone marrow chimeras cooperated with APC of either parental haplotype. This suggests that fetal liver and bone marrow may not be equivalent sources of stem cells. Our results may be due to fundamental differences between thymocyte precursors in fetal liver and bone marrow, including the time course of their expression of T cell receptor gene products.

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, trans-species evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relic of their unique historical demography, not their phylogenetic legacy.

We examined variation at MHC Class IIB genes in a recently established population of dark-eyed juncos (Junco hyemalis) in a coastal urban environment in southern California, USA relative to an ancestral-range population from a nearby species-typical montane environment. The founding population is estimated to have been quite small, but we predicted that variation at the major histocompatibility complex (MHC) among the founders would nevertheless be preserved owing to the high functional significance of MHC. Previous studies of MHC in songbirds have had varying degrees of success in isolating loci, as passerines show extensive MHC gene duplication. In order to compare diversity in the two populations, we employed two published approaches to sequencing MHC Class II exon 2: direct sequencing with exon-based primers, and traditional cloning and sequencing with intron-based primers. Results from both methods show that the colonist population has maintained high levels of variation. Our results also indicate varying numbers of alleles across individuals, corroborating evidence for gene duplication in songbird MHC. While future studies in songbirds may need to take a genomic approach to fully understand the structure of MHC in this lineage, our results show that it is possible to use traditional methods to reveal functional variation across populations. PMID:22685370

Two cyprinid species, Parachondrostoma toxostoma, an endemic threatened species, and Chondrostoma nasus, an invasive species, live in sympatry in southern France and form two sympatric zones where the presence of intergeneric hybrids is reported. To estimate the potential threat to endemic species linked to the introduction of invasive species, we focused on the DAB genes (functional MHC IIB genes) because of their adaptive significance and role in parasite resistance. More specifically, we investigated (1) the variability of MHC IIB genes, (2) the selection pattern shaping MHC polymorphism, and (3) the extent to which trans-species evolution and intergeneric hybridization affect MHC polymorphism. In sympatric areas, the native species has more diversified MHC IIB genes when compared to the invasive species, probably resulting from the different origins and dispersal of both species. A similar level of MHC polymorphism was found at population level in both species, suggesting similar mechanisms generating MHC diversity. In contrast, a higher number of DAB-like alleles per specimen were found in invasive species. Invasive species tended to express the alleles of two DAB lineages, whilst native species tended to express the alleles of only the DAB3 lineage. Hybrids have a pattern of MHCexpression intermediate between both species. Whilst positive selection acting on peptide binding sites (PBS) was demonstrated in both species, a slightly higher number of positively selected sites were identified in C. nasus, which could result from parasite-mediated selection. Bayesian clustering analysis revealed a similar pattern of structuring for the genetic variation when using microsatellites or the MHC approach. We confirmed the importance of trans-species evolution for MHC polymorphism. In addition, we demonstrated bidirectional gene flow for MHC IIB genes in sympatric areas. The positive significant correlation between MHC and microsatellites suggests that demographic

The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class IIMHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC. PMID:27588903

The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class IIMHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

The fundamental role of the major histocompatibility complex (MHC) in immune recognition has led to a general consensus that the characteristically high levels of functional polymorphism at MHC genes is maintained by balancing selection operating through host-parasite coevolution. However, the actual mechanism by which selection operates is unclear. Two hypotheses have been proposed: overdominance (or heterozygote superiority) and negative frequency-dependent selection. Evidence for these hypotheses was evaluated by examining MHC-parasite relationships in an island population of water voles (Arvicola terrestris). Generalized linear mixed models were used to examine whether individual variation at an MHC class II DRB locus explained variation in the individual burdens of five different parasites. MHC genotype explained a significant amount of variation in the burden of gamasid mites, fleas (Megabothris walkeri) and nymphs of sheep ticks (Ixodes ricinus). Additionally, MHC heterozygotes were simultaneously co-infected by fewer parasite types than homozygotes. In each case where an MHC-dependent effect on parasite burden was resolved, the heterozygote genotype was associated with fewer parasites, and the heterozygote outperformed each homozygote in two of three cases, suggesting an overall superiority against parasitism for MHC heterozygote genotypes. This is the first demonstration of MHC heterozygote superiority against multiple parasites in a natural population, a mechanism that could help maintain high levels of functional MHC genetic diversity in natural populations.

Allelic diversity at the major histocompatibility complex class II DP locus of rhesus macaques was studied by sequencing exon 2 of Mamu-DPA1 and -DPB1 genes. The Mamu-DPA1 gene is apparently invariant, whereas the Mamu-DPB1 locus displays polymorphism. Here we report the characterization of 1 Mamu-DPA1 and 13 Mamu-DPB1 alleles which were compared with other available primate Mhc-DPA1 and -DPB1 sequences. As compared with Mhc-DRB and -DQB1, most codons for the contact residues in the antigen binding site of the primate Mhc-DPB1 gene have a relatively low degree of variation in encoding various types of amino acids. In contrast to Mhc-DRB and -DQB, the HLA- and Mamu-DPB1 sequences cluster in a species-specific manner in phylogenetic trees. Mhc-DPB1 polymorphisms, however, are inherited in a transspecies mode of evolution, as is demonstrated by the sharing of lineage members between closely related macaque species. The data demonstrate that the transspecies character of Mhc-DPB1 polymorphism was retained over much shorter periods of time as compared with its sister class II loci, Mhc-DQ and -DR.

Abstracts Background Rhesus macaques living in western Sichuan, China, have been separated into several isolated populations due to habitat fragmentation. Previous studies based on the neutral or nearly neutral markers (mitochondrial DNA or microsatellites) showed high levels of genetic diversity and moderate genetic differentiation in the Sichuan rhesus macaques. Variation at the major histocompatibility complex (MHC) loci is widely accepted as being maintained by balancing selection, even with a low level of neutral variability in some species. However, in small and isolated or bottlenecked populations, balancing selection may be overwhelmed by genetic drift. To estimate microevolutionary forces acting on the isolated rhesus macaque populations, we examined genetic variation at Mhc-DQB1 loci in 119 wild rhesus macaques from five geographically isolated populations in western Sichuan, China, and compared the levels of MHC variation and differentiation among populations with that previously observed at neutral microsatellite markers. Results 23 Mamu-DQB1 alleles were identified in 119 rhesus macaques in western Sichuan, China. These macaques exhibited relatively high levels of genetic diversity at Mamu-DQB1. The Hanyuan population presented the highest genetic variation, whereas the Heishui population was the lowest. Analysis of molecular variance (AMOVA) and pairwise FST values showed moderate genetic differentiation occurring among the five populations at the Mhc-DQB1 locus. Non-synonymous substitutions occurred at a higher frequency than synonymous substitutions in the peptide binding region. Levels of MHC variation within rhesus macaque populations are concordant with microsatellite variation. On the phylogenetic tree for the rhesus and crab-eating macaques, extensive allele or allelic lineage sharing is observed betweenthe two species. Conclusions Phylogenetic analyses confirm the apparent trans-species model of evolution of the Mhc-DQB1 genes in these

MHC gene organization (size, complexity, gene order) differs markedly among different species, and yet all nonmammalian vertebrates examined to date have a true "class I region" with tight linkage of genes encoding the class I presenting and processing molecules. Three paralogous regions of the human genome contain sets of linked genes homologous to various loci in the MHC class I, class II, and/or class III regions, providing insight into the organization of the "proto MHC" before the emergence of the adaptive immune system in the jawed vertebrates.

The genes encoding major histocompatibility (MHC) molecules are among the most polymorphic genes known for vertebrates. Since MHC molecules play an important role in the induction of immune responses, the evolution of MHC polymorphism is often explained in terms of increased protection of hosts against pathogens. Two selective pressures that are thought to be involved are (1) selection favoring MHC heterozygous hosts, and (2) selection for rare MHC alleles by host-pathogen coevolution. We have developed a computer simulation of coevolving hosts and pathogens to study the relative impact of these two mechanisms on the evolution of MHC polymorphism. We found that heterozygote advantage per se is insufficient to explain the high degree of polymorphism at the MHC, even in very large host populations. Host-pathogen coevolution, on the other hand, can easily account for realistic polymorphisms of more than 50 alleles per MHC locus. Since evolving pathogens mainly evade presentation by the most common MHC alleles in the host population, they provide a selective pressure for a large variety of rare MHC alleles. Provided that the host population is sufficiently large, a large set of MHC alleles can persist over many host generations under host-pathogen coevolution, despite the fact that allele frequencies continuously change.

The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days. Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis. The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes. Images Figure 2 PMID:9135559

The prevalence and significance of precopulatory mate choice remains keenly debated. The major histocompatibility complex (MHC) plays a key role in vertebrate adaptive immunity, and variation at the MHC influences individual survival. Although MHC-dependent mate choice has been documented in certain species, many other studies find no such pattern. This may be, at least in part, because in natural systems constraints may reduce the choices available to individuals and prevent full expression of underlying preferences. We used translocations to previously unoccupied islands to experimentally reduce constraints on female social mate choice in the Seychelles warbler (Acrocephalus sechellensis), a species in which patterns of MHC-dependent extrapair paternity (EPP), but not social mate choice, have been observed. We find no evidence of MHC-dependent social mate choice in the new populations. Instead, we find that older males and males with more microsatellite heterozygosity are more likely to have successfully paired. Our data cannot resolve whether these patterns in pairing were due to male–male competition or female choice. However, our research does suggest that female Seychelles warblers do not choose social mates using MHC class I to increase fitness. It may also indicate that the MHC-dependent EPP observed in the source population is probably due to mechanisms other than female precopulatory mate choice based on MHC cues. PMID:26792973

Purpose: Topoisomerase II{alpha} (Topo II{alpha}) plays a role in DNA replication and is the molecular target for anthracyline-based chemotherapy. The purpose of this study was to evaluate the relationship between Topo II{alpha} expression and survival in patients with invasive breast cancer. Methods and Materials: Formalin-fixed, paraffin-embedded tumor specimens from 24 women with invasive breast cancer were stained for Topo II{alpha} expression. All women underwent mastectomy. Radiotherapy was given at University of Utah Department of Radiation Oncology. Of the patients, 23 (96%) received chemotherapy. The level of Topo II{alpha} expression within tumor cells was compared with clinical factors and overall survival. Results: The median percentage of tumor cells expressing Topo II{alpha} was 70%. Increased Topo II{alpha} tumor expression significantly correlated with diminished disease-free survival. Five-year disease-free survival was 100% for patients with <70% of breast cancer cells expressing Topo II{alpha} compared with 42% for patients with {>=}70% Topo II{alpha} expression (p 0.008). The level of Topo II{alpha} expression within tumor cells correlated with T stage (p = 0.008) but not with other pathologic factors. Conclusions: Increased Topo II{alpha} expression significantly correlated with diminished disease-free survival in patients with invasive breast cancer. These findings may indicate a role for Topo II{alpha} expression as a prognostic factor in breast cancer.

A functional immune system requires a tight control over major histocompatibility complex (MHC) gene transcription, as the abnormal MHCexpression patterns of severe immunodeficiency and autoimmune diseases demonstrate. Although the regulation of MHCexpression has been well documented in humans and mice, little is known in other species. In this study, we detail the level of polymorphism in wolf and dog MHC gene promoters. The promoter regions of the DRB, DQA and DQB locus were sequenced in 90 wolves and 90 dogs. The level of polymorphism was high in the DQB promoters, with variation found within functionally relevant regions, including binding sites for transcription factors. Clear associations between DQB promoters and exon 2 alleles were noted in wolves, indicating strong linkage disequilibrium in this region. Low levels of polymorphism were found within the DRB and DQA promoter regions. However, a variable site was identified within the T box, a TNF-alpha response element, of the DQA promoter. Furthermore, we identified a previously unrecognised 18-base-pair deletion within exon 1 of the DQB locus.

The fundamental role of the major histocompatibility complex (MHC) in immune recognition has led to a general consensus that the characteristically high levels of functional polymorphism at MHC genes is maintained by balancing selection operating through host–parasite coevolution. However, the actual mechanism by which selection operates is unclear. Two hypotheses have been proposed: overdominance (or heterozygote superiority) and negative frequency-dependent selection. Evidence for these hypotheses was evaluated by examining MHC–parasite relationships in an island population of water voles (Arvicola terrestris). Generalized linear mixed models were used to examine whether individual variation at an MHC class II DRB locus explained variation in the individual burdens of five different parasites. MHC genotype explained a significant amount of variation in the burden of gamasid mites, fleas (Megabothris walkeri) and nymphs of sheep ticks (Ixodes ricinus). Additionally, MHC heterozygotes were simultaneously co-infected by fewer parasite types than homozygotes. In each case where an MHC-dependent effect on parasite burden was resolved, the heterozygote genotype was associated with fewer parasites, and the heterozygote outperformed each homozygote in two of three cases, suggesting an overall superiority against parasitism for MHC heterozygote genotypes. This is the first demonstration of MHC heterozygote superiority against multiple parasites in a natural population, a mechanism that could help maintain high levels of functional MHC genetic diversity in natural populations. PMID:19129114

A variety of nonrandom mate choice strategies, including disassortative mating, are used by vertebrate species to avoid inbreeding, maintain heterozygosity and increase fitness. Disassortative mating may be mediated by the major histocompatibility complex (MHC), an important gene cluster controlling immune responses to pathogens. We investigated the patterns of mate choice in 26 wild-living breeding pairs of gray wolf (Canis lupus) that were identified through noninvasive genetic methods and genotyped at 3 MHC class II and 12 autosomal microsatellite (STR) loci. We tested for deviations from random mating and evaluated the covariance of genetic variables at functional and STR markers with fitness proxies deduced from pedigree reconstructions. Results did not show evidences of MHC-based disassortative mating. Rather we found a higher peptide similarity between mates at MHC loci as compared with random expectations. Fitness values were positively correlated with heterozygosity of the breeders at both MHC and STR loci, whereas they decreased with relatedness at STRs. These findings may indicate fitness advantages for breeders that, while avoiding highly related mates, are more similar at the MHC and have high levels of heterozygosity overall. Such a pattern of MHC-assortative mating may reflect local coadaptation of the breeders, while a reduction in genetic diversity may be balanced by heterozygote advantages.